Suppressor of HIV replication and transcription

ABSTRACT

The present invention relates to a bioactive molecule, herein referred to as the CD8 +  suppressor molecule, that is produced by the CD8 +  subset of human T-lymphocytes and suppresses type-1 human immunodeficiency virus (HIV-1) replication through inhibition of viral transcription. The invention relates to isolation of CD8 +  cell lines and cell clones that produce that antiviral activity and to the development of assay systems for detection of the antiviral activity. The cell lines, cell clones and assay systems, described herein, may be utilized, e.g., to purify, characterize and clone the CD8 +  suppressor molecule. The CD8 +  suppressor molecule may have therapeutic applications for treatment of diseases associated with HIV-1 infection.

1. INTRODUCTION

[0001] The present invention relates to bioactive molecules, hereinreferred to as CD8⁺ suppressor molecules, that are produced by the CD8⁺subset of human T-lymphocytes and suppress human immunodeficiency virus(HIV) replication through inhibition of viral transcription. Theinvention relates to isolation of clonal CD8⁺ cell lines and/or thegeneration of permanently established transformed CD8⁺ cell lines thatproduce antiviral activity. The invention also relates to thedevelopment of assay systems for detection of the antiviral activity.The cell lines and assay systems, described herein, may be utilized,e.g., to purify, characterize and clone a CD8⁺ suppressor molecule. TheCD8⁺ suppressor molecules may be used, e.g., in therapeutic applicationsfor treatment of diseases associated with HIV infection.

2. BACKGROUND OF THE INVENTION

[0002] The type-1 human immunodeficiency virus (HIV-1) has beenimplicated as the primary cause of the slowly degenerate disease of theimmune system termed acquired immune deficiency syndrome (AIDS)(Barré-Sinoussi, F. et al., 1983 Science 220:868-70; Gallo, R. et al.1984, Science 224:500-3). Infection of the CD4⁺ subclass ofT-lymphocytes with the HIV-1 virus leads to depletion of this essentiallymphocyte subclass which inevitably leads to opportunistic infections,neurological disease, neoplastic growth and eventually death. HIV-1infection and HIV-1 associated diseases represent a major health problemand considerable attention is currently being directed towards thesuccessful design of effective therapeutics.

[0003] HIV-1 is a member of the lentivirus family of retroviruses(Teich, N. et al., 1984 In RNA Tumor Viruses ed. R. Weiss, N. Teich, H.Varmus, J. Coffin CSH Press, pp. 949-56). The life cycle of HIV-1 ischaracterized by a period of proviral latency followed by activereplication of the virus. The primary cellular target for the infectiousHIV-1 virus is the CD4⁺ subset of human T-lymphocytes. Targeting of thevirus to the CD4⁺ subset of cells is due to the fact that the CD4⁺ cellsurface protein acts as the cellular receptor for the HIV-1 virus(Dalgleish, A. et al., 1984, Nature 312:763-67; Klatzmann et al. 1984,Nature 312:767-68; Maddon et al. 1986 Cell 47:333-48).

[0004] In more detail, HIV-1 infection of susceptible cells is initiatedvia interactions between the virus envelope glycoprotein (gp120) and theCD4⁺ cell surface receptor. Fusion of the viral and cell membranes thenproceeds through subsequent interaction of this complex with a specificchemokine receptor, primarily the CCR5 or the CXCR4 chemokine receptor(Bieniasz & Cullen, 1998, Front. Biosci. 3:D44-D58; Moore et al., 1997,Curr. Opin. Immunol. 9:551-562). HIV-1 isolates that can infect T-celllines and induce syncytia (SI) use the CXCR4 receptor and are termed X4HIV-1. Such isolates are typically recovered late in the course of HIVprogression and differ from the non-syncytia inducing (NSI) strainswhich predominate in the early stages of HIV infection. NSI strains gainentry to target cells through use of the CCR5 receptor and are referredto as R5 HIV-1.

[0005] After binding to the cell surface and fusion of the virus andcell membrane, the HIV-1 virion becomes internalized and the virus's RNAgenome is converted into linear double-stranded DNA molecules. Thisprocess is dependent on the action of the virally encoded reversetranscriptase. Following replication of the viral genome, the linear DNAmolecule integrates into the host genome through the action of the viralintegrase protein, thus establishing the proviral form of HIV-1. Duringthe early phase of proviral expression, transcription of the viralgenome results in expression of regulatory proteins such as Tat, Nef andRev. Transcriptional activation of the proviral DNA is mediated throughthe viral 5′ LTR sequences (long terminal repeats). The initial lowlevel of viral transcription is dramatically increased by the HIVencoded transactivator protein termed tat (transactivator protein)(Cullen, B. R. et al. 1989, Cell 58:423-26). The Rev protein promotesthe transition from the early phase expression of regulatory proteins tolate phase expression of structural proteins. Assembly of newlysynthesized viral particles is followed by budding of virus particlesfrom the cell membrane allowing the virus to infect new cells.

[0006] The HIV-1 virus is capable of establishing a latent state ofinfection for prolonged periods of time. Individuals infected with thehuman immunodeficiency virus may remain clinically healthy for longperiods of time, with the estimated average length of the asymptomaticperiod between primary HIV infection and the progression to AIDS andincrease in viral replication being approximately 8 to 10 years. It isgenerally believed that the humoral immune response to HIV-1 is notsufficiently protective against progression of the disease. Therefore,attention has turned to the possibility that the T-lymphocyte populationof cells may maintain the period of latency by directly inhibiting HIV-1replication.

[0007] A number of groups have recently noted that the CD8⁺ subset ofT-lymphocytes have the ability to inhibit the replication of HIV-1 invitro (Walker, C. M. et al., 1989, Cellular Immunology 119:470-475;Kannagi, M. et al. 1990, J. Virology 64:3399-3406; Walker, C. M. et al.,1991 J. Virology 65:5921-5927). For example, addition of CD8⁺ cells tonaturally HIV-1 infected CD4⁺ cell cultures has been found to inhibitthe replication of HIV-1 in the infected cultures in a dose dependentmanner. (Ref. supra). Furthermore, the inhibitory effect is not entirelydependent on cell-cell contact as an inhibitory effect is observedacross a semi-permeable membrane suggesting that at least a portion ofthe CD8⁺ suppressor activity is due to a soluble inhibitor of HIV-1replication. (Ref. supra). To date, the molecular identity of the CD8⁺suppressor molecule, or a combination of factors, as well as themechanism by which it exerts its antiviral effect remains undefined.

[0008] Because such CD8⁺ suppressor molecules would be useful, e.g., inthe treatment and/or inhibition of HIV infection, there is a need in theart to identify and characterize such molecules and their HIVsuppression activity. There is also a need for methods, particularlyscreening methods, which may be used, e.g., to screen CD8⁺ cells forsuch suppression activity and to isolate such CD8⁺ suppressor molecules.

3. SUMMARY OF THE INVENTION

[0009] The present invention relates to molecules produced by the CD8⁺subclass of T-lymphocytes that are able to inhibit HIV viralreplication. The molecules can be e.g., soluble molecules producedand/or secreted by the CD8⁺ subclass of T-lymphocytes or alternatively,can be expressed on the surface of a CD8⁺ lymphocyte. The inventionfurther relates to the observation that the mechanism by which thesuppressor molecule exerts its antiviral activity is at the level ofinhibition of viral gene expression from the viral LTR promoter.

[0010] The invention also relates to and is based, at least in part, onthe discovery that the antiviral activity of a CD8⁺ suppressor moleculeoccurs at a stage or stages of the viral replication cycle after viralentry into CD4⁺ cells and before the stages of expression of late geneproducts, viral assembly, maturation and budding. For example, theExample presented in Section 10, below, presents data, for the firsttime, demonstrating that CD8⁺ suppressor activity occurs during a stageof the viral replication cycle such as integration of viral DNA,transactivation from the proviral state, export of tat and/or rev intothe cytoplasm and then back into the nucleus and/or tat mediatedenhancement of transcription.

[0011] In a principle embodiment, the invention is directed to an assaysystem that can be used for detection of HIV inhibitory activity duringa single cycle of HIV infection. In particular, the invention provides asingle cycle HIV-1 reporter vector wherein a reporter gene isoperatively associated with an HIV-1 promoter. In preferred embodimentsof the invention, the reporter gene is expressed during the earlyproviral gene expression stage of the viral replication cycle. Forexample, in a particularly preferred embodiment described herein, thereporter gene is expressed in place of one or more early proviral genes,such as in place of the HIV-1 nef gene.

[0012] In such an assay system, the presence of a suppressor moleculecan be detected by measuring the levels of the reporter gene product.Such an assay system can also be used to identify the stage in theproviral replication cycle targeted by the suppressor molecule. Thus,the assay system enables a user to both detect a suppressor molecule andcharacterize its activity.

[0013] Using such a pseudotyped virus screening assay a user can readilyscreen for and detect suppressor compounds, including CD8⁺ suppressorcompounds, that inhibit HIV replication during a particular stage orparticular stages of the HIV replication cycle. For example, compoundscan be detected and/or identified which inhibit HIV by targeting thestage of viral entry, the stage of reverse transcription or the stage ofearly viral gene expression, to name a few. The method simply involvescontacting a host cell with a single cycle HIV pseudotyped virus of theinvention followed by contacting the host cell with a test compound. HIVinhibition is then ascertained by detecting inhibition of reporter geneactivity. An HIV suppressor compound will inhibit a single cycle of HIVreplication as long as it is contacted to the host cell prior to a timeinterval when the stage of the pseudotyped virus replication cycletargeted by the compound is complete (i.e., after viral entry, afterreverse transcription or after expression of early viral genes).

[0014] The invention also provides an assay system, which is alsodescribed in U.S. Pat. Nos. 5,627,023 and 5,861,490 (filed on Mar. 29,1993 and Jun. 6, 1995, respectively) to be used for detection of the HIVinhibitory activity, whereby the HIV LTR sequence is cloned adjacent toa reporter gene such as the CAT gene. In such an assay system, thepresence of the suppressor molecule may be determined by measuring thelevels of reporter gene product.

[0015] The present invention further relates to the isolation andcharacterization of CD8⁺ cell clones that produces the antiviralactivity, e.g., using one or more of the screening assays of theinvention. The invention also relates to the generation of permanentlyestablished CD8⁺ cell lines that produce the antiviral activity. Theantiviral activity produced by such cell lines may be produced by asoluble, cytolytic molecule produced by said cell lines or,alternatively, may be produced by a non-cytolytic molecule produced bysaid cell lines (e.g., a molecule expressed on the membrane of said celllines). Such cell lines may be generated by transferring cellular orviral genes capable of transformation or immortalization into CD8⁺cells. Exemplary cell lines which are considered part of the presentinvention include the cell lines described hereinbelow, which referredto as DU-JR.HVS and DU.HS-HVS and identified by the ATCC Accession Nos.(### ##### and ### #####, respectively).

[0016] CD8⁺ clonal cells and/or CD8⁺ permanently established cell linesthat produce the antiviral activity of interest may be advantageouslyused for large scale isolation and characterization of the suppressormolecule and/or as a source of mRNA for construction of cDNA librariesthat may be used for cloning the suppressor molecule. The invention alsorelates to the use of the suppressor molecule in the treatment ofHIV-infection.

4. BRIEF DESCRIPTION OF THE FIGURES.

[0017]FIG. 1. CD8⁺ Cells from HIV-1 Seropositive Individuals Suppressesvirus production.

[0018]FIG. 2. Plasmid constructs used in HIV-1 LTR transcription assays.

[0019]FIG. 3. CD8⁺ Cells from HIV-1 infected individuals suppress HIV-1LTR transcription. Data are plotted for CAT activity in culturescontaining autologous CD8⁺ cells compared to the activity measured incultures derived from the same transfection containing autologous CD4⁺cells. Horizontal lines are drawn to indicate the means of eachpopulation.

[0020]FIG. 4. CD8⁺ cells suppress Tat-mediated transcription inheterologous CD4⁺ cells. Data are plotted for CAT activity in culturescontaining autologous or heterologous CD8⁺ cells compared to theactivity measured in cultures derived from the same transfectioncontaining autologous CD4⁺ cells.

[0021]FIG. 5. A soluble factor from CD8⁺ cells inhibits Tat-mediatedtranscription. CAT activity was measured in cultures containingautologous CD4⁺ cells, cultures containing autologous CD8⁺ cells andcultures containing autologous CD8⁺ cell conditioned medium. Each dataset from an individual subject was derived from a single transfection.CAT activity is expressed as percent conversion, each assay was based on5×10⁶ transfected CD4⁺ cells.

[0022]FIG. 6. HIV transcriptional inhibition is expressed by a primaryCD8⁺ cell clone.

[0023]FIG. 7. Anti-HIV-1 activity of CD8⁺ clones in autologous andheterologous co-culture screening assays. Controls represent RTproduction of infected CD4⁺ cells in the absence of other cells. (A)Clones from patient S were screened against both heterologous, acutelyinfected CD4⁺ cells and autologous, naturally infected CD4⁺ cells. Thedata shown was collected on day 6 of assay. (B) Clones from patient Bwere screened against autologous, naturally infected CD4⁺ cells. Datashown was also collected on day 6. (C) Clones from patient R werescreened against heterologous, naturally infected CD4⁺ cells. Data shownwas collected on day 3 of assay.

[0024]FIG. 8. Autoradiograph of RT assay showing anti-HIV-1 activity ofclone B11 compared to non-active clone B18 and to acutely infected CD4⁺cells alone. The E:T ratio at the commencement of the assay was 2:1.

[0025]FIG. 9. CD8⁺ clones from Patient B were separated from acutelyinfected CD4⁺ targets by a filter with 0.4 μm pores in the context oftranswell plates (Costar). A 4:1 E:T ratio was employed. Supernatant wassampled for RT activity at day 4, 6, and 8 after culture as shown. Thesecond graph shows the results of a parallel coculture experiment. A 2:1E:T ratio was employed. The effectors of both experiments originatedfrom the same stock culture.

[0026]FIG. 10. Establishment of a HVS-transforned CD8⁺ population. CD8⁺cells from three HIV⁺ patients 1(□), 2(♦), and 3(∘) were exposed to HVSat day 0. Viable cell numbers as measured by erythrosin red dyeexclusion are shown over the course of 57 days.

[0027]FIG. 11. IL-2-dependence of the HVS-transformed CD8⁺ cells. Thecells were cultured in the presence of 0(▪), 5(♦), 25(), and 100(▴)units of recombinant IL2/ml of AIM-V medium unsupplemented with fetalbovine serum (FBS). Viable cell numbers were ascertained at dailyintervals.

[0028]FIG. 12. Detection of HVS DNA sequences in the transformed bulkCD8⁺ population using the polymerase chain reaction. In the upper panelprimers corresponding to the HVS dihydrofolate reductase genes wereused. In the lower panel the primers were specific for the HVS ORF 2.

[0029]FIG. 13. PCR amplification of the Vβ region of the T-cell receptorgenes in the bulk HVS-transformed CD8⁺ cells from a patient.

[0030]FIG. 14. (A) Comparison of the ability of primary andHVS-transformed CD8⁺ cells to suppress HIV-1 production by CD4⁺ cellsfrom the same patient. The figures represent autoradiographs of DE81membrane-bound RT assay products corresponding to cell culturesupernatants. (B) HVS-transformed CD8⁺ cells can suppress HIV-1production by CD4⁺ cells from a MHC class I (MHC-I) mismatched patient.

[0031]FIG. 15. Comparison of HVS-transformed CD8⁺ cell inhibition ofvirus production in transwell and cocultivation assays. (A) A CD4⁺-CD8⁺cocultivation assay was performed in parallel using the samepreparations of cells utilized for the transwell study. (B) Transwellexperiment using HIV⁺ CD4⁺ cells in the upper compartment andHVS-transformed CD8⁺ cells in the lower compartment at varying ratios tothe CD4⁺ cells. The cultures were sampled at intervals between days 6and 17. Each point in the cocultivation assay is the average of 3replicate culture wells.

[0032]FIG. 16. Data from a quantitative CD8⁺ suppression assay performedwith both a CCR5-dependent or “R5” HIV isolate (QZ4734) and a CXCR4dependent or “X4” HIV isolate (IIIB) in a 96 well tissue culture plateformat using CD4⁺ enriched cells as the target.

[0033]FIG. 17. HIV suppressor activity seen in CD4⁺ T-lymphocyte cellsinfectected with a CXCR-4 dependent pseudotyped virus (NL4-3) withoutCD8⁺ cells or with CD8⁺ T-lymphocyte cells from both HIV-positive andHIV-negative individuals; cells were combined at various ratios of CD8⁺cells to CD4⁺ cells (i.e., at various “effector:target” or “E:T”ratios).

[0034]FIG. 18. Data from time of addition experiments detecting HIVsuppression in CD4⁺ T-lymphocytes infected with a CXCR-4 dependentpseudotyped virus (NL4-3); (A) CD8⁺ T-lymphocytes derived fromasymptomatic HIV-positive individuals were added to the CD4⁺ cells atvarious times after exposure to the pseudotyped virus; (B) the viralentry inhibitors DP178 and anti-CD4⁺ monoclonal antibody (mAb#19) wereadded to the CD4⁺ cells at various times after exposure to thepseudotyped virus.

[0035]FIG. 19. Data from time of addition experiments detecting HIVsuppression in CD4⁺ T-lymphocytes infected with a CXCR4 dependentpseudotyped virus (NL4-3); the following viral inhibitors were added atvarious times after exposure to the psuedotype virus: DP178 (20 μg/ml),nevirapine (0.2 μM), nelfinavir (0.2 μM) and Tat inhibitor (50 μM).

5. DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention relates to molecules produced by the CD8⁺subclass of T-lymphocytes that are able to inhibit HIV viralreplication. The molecules can be e.g., soluble molecules producedand/or secreted by the CD8⁺ subclass of T-lymphocytes or alternatively,can be expressed on the surface of a CD8⁺ lymphocyte. The inventionfurther relates to an assay system that can be used for detection of HIVinhibitory activity during a single cycle of HIV infection. Inparticular, the invention provides a replication deficient HIVpseudotyped virus comprising a reporter vector having a reporter genethat is expressed during early proviral gene expression. In such anassay system, the presence of a suppressor molecule can be detected bymeasuring the levels of the reporter gene product. As the term is usedherein, a “replication deficient HIV pseudotyped virus” refers,specifically, to an HIV pseudotyped virus that is unable to complete areplication cycle. Thus, the replication deficient pseudotyped virusesof the present invention are characterized by their ability to undergo,at most, a single cycle of infection or, alternatively, have the abilityto undergo only a portion of a single cycle of HIV infection.

[0037] Such an assay system can also be used to identify the stage orstages in the viral replication cycle that are targeted by a suppressormolecule. In a particularly preferred embodiment, the assay system canbe used to identify steps up to and including expression of earlyproviral genes. For example, the Example presented in Section 10, below,demonstrates for the first time, using the assay system of the presentinvention, that CD8⁺ suppressor activity targets HIV at a stage of theviral replication cycle after entry of the virus in a cell (e.g., afterentry into a CD4⁺ cell). Thus, the assay system enables a user to bothdetect a suppressor molecule and characterize its activity.

[0038] The invention also provides an assay system for detection of theantiviral activity wherein a reporter gene is cloned adjacent to the HIVviral LTR sequences. The development of the assay system is based on theobservation that the suppressor molecule inhibits transcription of geneslinked to the HIV LTR promoter sequences. In addition, the invention isdirected to the isolation of clonal CD8⁺ cells that exhibit theantiviral activity (i.e., CD8⁺ cells that express an HIV suppressormolecule). The invention is further related to the generation ofpermanently established CD8⁺ cell lines that express the antiviralactivity. Such established cell lines are characterized by their abilityto proliferate continuously in tissue culture. Such cell lines may beadvantageously used for purification and characterization of thesuppressor molecule and/or for cloning of the CD8⁺ suppressor molecule.The CD8⁺ suppressor molecule may be used therapeutically to inhibitHIV-replication.

5.1. The CD8⁺ Suppressor Molecule Inhibits HIV-1 Viral Transcription

[0039] The effect of CD8⁺ cells on HIV-1 replication was investigated byperforming experiments in which CD8⁺ cells, prepared from HIV-1 infectedindividuals by immunoaffinity techniques, were mixed with virallyinfected CD4⁺ cells in a 2:1 ratio. The inhibition of HIV-1 viralreplication can be measured by determining the levels of viral reversetranscriptase activity produced by HIV-1 infected cells. As illustratedin FIG. 1, inhibition of viral replication was virtually complete in thepresence of CD8⁺ cells.

[0040] Experiments, described in the Example presented in Section 6,below, and in U.S. Pat. Nos. 5,627,023 and 5,861,490 filed on Mar. 29,1993 and Jun. 6, 1995, respectively, were performed to test whether themechanism by which the CD8⁺ suppressor molecule inhibits viralreplication is through inhibition of viral gene transcription. Arecombinant expression vector was constructed comprising the HIV-1 LTRpromoter sequences cloned adjacent to the CAT reporter gene (FIG. 2).The construct was co-transfected into affinity purified CD4⁺ cells witha second construct expressing the product of the viral Tat gene which isrequired for viral transcription. A construct consisting of the CAT genecloned adjacent to the cytomegalovirus immediate early promoter (CMV-IE,FIG. 2) was used as a control (FIG. 2). As indicated in FIG. 3,decreased levels of CAT activity were observed in the presence ofautologous CD8⁺ cells, indicating inhibition of HIV-1 LTR andTat-mediated HIV-1 LTR transcription in the autologous CD4⁺ infectedcells. Similar experiments were carried out using heterologous CD8⁺cells. When these cells were mixed with the transfected CD4⁺ cells adecrease in CAT activity was also observed (FIG. 4) indicating thatcompatibility at the major histocompatibility locus (MHC) is notrequired for HIV-1 suppressor activity. In addition, supernatantsderived from CD8⁺ cell culture exhibited inhibitory activity indicatingthat at least a portion of the antiviral activity is a soluble factorsecreted by the CD8⁺ cells (FIG. 5).

[0041] Additional experiments, which are described for the first time inthe example presented in Section 10, below, were performed to moreprecisely identify the stage or stages of the viral replication cyclewhich are targeted by CD8⁺ suppressor activity. The experiments utilizean assay that detects HIV inhibitory activity during a single cycle ofHIV infection. The experiments were done using a replication deficientHIV pseudotyped virus comprising a reporter vector having a reportergene that is operatively associated with the HIV-1 promoter and isexpressed in place of an early proviral gene. Such an early proviralgene may be, for example, the HIV nef tat, or rev gene, to name a few.In a particularly preferred embodiment, which was used in theexperiments described in Section 10, below, the early proviral gene isthe HIV nef gene. Although not limiting of the present invention in anyway, the particular, preferred reporter vector used in the experimentsdescribed below in Section 10 comprises the reporter virus DNA pNL4-3LUCR⁻ E⁻—an Env deficient construct containing a luciferase reportergene in the place of the viral nef gene. Such constructs are known inthe art and have been described, e.g., in Connor et al., 1995, Virology206:935-944 and in Chen et al., 1994, J. Virol. 68:654-660. Theconstruct was co-transfected into human embryonic kidney cells with asecond construct pNL4-3 env, which expresses the product of the viralEnv gene. A second pseudotyped virus comprising the reporter virus DNAco-transfected with an expression vector for the amphotropic murineleukemia virus envelope, A-MLV (S-A-MLV-env) was used in an assay todemonstrate that the observed HIV suppressor activity is unrelated tothe pseudotyped virus's envelope protein. Thus, the pseudotyped virusesof the invention are not limited to pseudotyped viruses having an HIVEnv coat protein, but also include pseudotyped viruses having other, nonHIV, protein coats. Such pseudotyped viruses can be readily produced byco-transfecting DNA for such a pseudotyped virus with a vector thatencodes for another viral coat protein. For example, a pseudotyped virusused in the methods of the present invention can have a viral proteincoat corresponding to the protein coat of murine leukemia virus,vesicular stomatitis virus G (VSV-G) or ebola virus glycoprotein(Ebo-GP), to name a few. Likewise, the pseudotyped viruses used in themethods of the present invention can have a viral coat proteincorresponding to the protein coat of any strain of HIV. For example, apseudotyped virus of the invention can have an Env coat protein of an X4HIV-1 virus (i.e., an HIV strain that infects T-cells using the CXCR4receptor). Alternatively, a pseudotyped virus of the invention can havean Env coat protein of an R5 HIV-1 virus (i.e., an HIV strain thatinfects T-cells using the CCR5 receptor).

[0042] The resulting pseudotyped viruses are capable of infecting (i.e.,entering) appropriate host cell and undergo reverse transcription,translocation to the nucleus, integration and expression of early virusgenes, including Tat, which then acts to upregulate expression of theluciferase reporter. For example, in preferred embodiments wherein thevirus has been pseudotyped with an HIV-1 Env protein, the pseudotypedvirus is capable of infecting affinity purified CD4⁺ cells. Thus, thesystem recapitulates the viral replication process up to and includingexpression of early virus genes.

[0043] As indicated in FIG. 17, decreased levels of luciferase activitywere observed in the presence of CD8⁺ cells, indicating inhibition ofHIV-1 replication before or during the stage of early virus geneexpression in infected CD4⁺ cells. Similar experiments were conductedusing CD8⁺ cells added to CD4⁺ cells at various times after exposure tothe provirus (FIG. 18A) indicating that CD8⁺ suppression of HIVreplication occurs as late as 24 hours post viral exposure. By comparingdata from similar experiments using other HIV inhibitors, the stage orstages of the virus replication cycle that are inhibited by CD8⁺suppressor molecules could be more precisely identified. For example,time of addition assays with viral entry inhibitors (DP178 and anti-CD4⁺mAb) indicate that these inhibitors suppress pseudotyped virusreplication within 2-6 hours post virus exposure (FIGS. 18B and 19). Bycontrast, a reverse transcription inhibitor (nevirapine) suppressesreplication within approximately 10 hours post exposure and an inhibitorof early virus gene expression (Tat inhibitor) suppresses pseudotypedvirus replication if added as late as 24 hours post exposure (FIG. 19).A protease inhibitor, however, targets a stage of the virus replicationcycle after early virus gene expression and does not, therefore, inhibitpseudotyped virus expression in these assays (FIG. 19).

[0044] These data demonstrate, therefore, that a target inhibited by aCD8⁺ suppressor molecule is one which is active during the latest stagesof the pseudotyped virus's life cycle. Such targets including, but arenot limited to, integration of viral DNA, transactivation from theproviral state, export of Tat and Rev into the cytoplasm and then backinto the nucleus and/or Tat mediated enhancement of transcription. Inparticular, the data presented herein below demonstrates that such CD8⁺suppressor activity is not mediated by a compound, such as abeta-chemokine, which inhibits viral entry.

[0045] In addition, other viruses of human and animal origin containsimilar and/or identical promoter elements as those found in HIV,raising the possibility that the CD8⁺ suppressor molecule may be ofvalue in the treatment of other viral infections such as HIV-2, HTLV-1and 2, FeLV, etc.

5.2. Subsets of CD8⁺ Cells Express Anti-Viral Activity

[0046] The availability of CD8⁺ cell clones expressing antiviralactivity will permit extensive surface marker phenotyping of the cellsproducing this activity. Once the distinguishing phenotype(s) of thesecells is established, improved immunoaffinity techniques for purifyingthese cells can be devised. If these cells play a role in maintainingthe asymptomatic state during HIV infection, these markers may be usefulfor a) clinical staging of infected individuals, b) monitoring theeffect of antiviral therapy on disease progression, c) monitoring theeffectiveness of therapy with immunological/biological responsemodifiers, and d) monitoring vaccine response.

[0047] Such cell lines may also be used for purification andcharacterization of the suppressor molecule using methods and techniquesdescribed in Section 5.3., infra. The cell lines may also be used as asource of RNA from which cDNA libraries may be constructed as describedin Section 5.4., infra.

[0048] In a specific embodiment, described herein, CD8⁺ cells wereimmunoaffinity purified from the blood of an HIV infected patient andthe purified CD8⁺ cells were subjected to limited dilution cloning. Theresulting CD8⁺ primary cell clones were assessed for their ability toinhibit tat-mediated HIV LTR transcription in autologolous B-lymphocytescell lines transfected with the tat encoding vector and the LTR/CATexpression vector. FIG. 6 shows the level of CAT inhibition observed forthree CD8⁺ primary cell clones. As indicated in the Figure, the clonesexhibit varying degrees of inhibition of transcription. Clone 2 (DU.HL-1) shows the greatest inhibition of CAT activity while clone 4 (DU.HL-4) does not inhibit activity.

[0049] In alternative preferred embodiments, CD8⁺ primary clones can beassessed for their ability to inhibit replication of a single cyclereporter vector such as the pseudotyped virus reporter vector describedin Section 5.1, above, and used in the experiments presented in Section10, below. For example, such an assay may be used to screen and identifyCD8⁺ primary clones that suppress pseudotyped virus replication at aparticular stage or stages of the virus replication cycle. In onepreferred embodiment, such a screening assay can be used to screen forand identify clones that suppress pseudotyped virus replication duringthe latest stages of pseudotyped virus replication including, but notlimited to, during integration of viral DNA, transactivation from theproviral state, export of Tat and/or Rev into the cytoplasm and/or backinto the nucleus and/or Tat mediated enhancement of transcription.

[0050] As lymphocytes proliferate and differentiate they express cellsurface markers which may be used to identify the different classes oflymphocytes present in the body. The different classes of lymphocytesmay be sorted based on the differential expression of cell surfacemarkers using antibodies directed to those cell surface markers and flowcytometry. In yet another embodiment of the invention, described herein,flow cytometry was used to measure cell surface markers expressed on thecell surface of clonal populations of CD8⁺ cells derived fromasymptomatic HIV-1 infected patients. The clonal cell lines from each ofthe individual patients varied in their ability to inhibit HIVreplication. As indicated in TABLE I a variety of phenotypic markers aredisplayed within the suppressive and non-suppressive clonal populations.The suppressive clones tended to express activation markers such asHLA-DR, S6FI, CD25 and CD28 to a much higher degree than non-suppressiveclones. TABLE I Clone Phenotypic Non-Suppressive Suppressive Marker B5.5B18 B6 B11 B22.5 S92 HLA-DR 14% 25% 13% 82% 47% 72% CD25  4% 17% 58% 58%16% 40% CD38  4% 65% 67% 90% 61% 76% CD28  0% 41% 64% 35% 37% 16% CD45RAND  5%  4% 19% ND 34% CD45RO ND 45% 67% 82% ND 65% S6F1 23% 16% 14% 59%56% 57% CD57 ND  0%  2%  0% ND 10% TcR (Vβ)  5.1 1  5.1 12    ND 13   

[0051] The increased expression of specific cell surface markers on CD8⁺cells expressing antiviral activity will permit the use of improvedimmunoaffinity techniques for purifying these cells.

5.3. Generation of CD8⁺ Permanently Established Cell Lines That ExpressAnti-Viral Activity

[0052] Cultures of normal lymphocytes tend to stop growing after afinite number of generations. Permanent lymphocyte cell lines may begenerated by the transfer of genetic information encoding cellular orvirally derived oncogenes. The transfer and stable uptake of suchoncogenes into the genome of purified lymphocytes will confer on thosetransformed cells the ability to proliferate continuously in tissueculture.

[0053] In an embodiment of the invention permanent CD8⁺ cells lines weregenerated by preparing CD8⁺ cells from HIV⁺ patients and exposing thepurified CD8⁺ cells to Herpesvirus saimiri. One particular transformedculture, acquired from patient 2 and designated DU.WS-1-CD8 (HVS), waschosen for further analysis. As demonstrated in FIG. 12, DU.WS-1-CD8(HVS) cells contained HSV DNA integrated into their genome as indicatedby detection of polymerase chain reaction products of the predictedsize. In addition, as presented in FIG. 14 the transformed CD8⁺ cellswere as potent as primary CD8⁺ cells in their ability to inhibit HIV-1replication in HIV-infected CD4⁺ cells. The results presented in FIG. 15demonstrate that the transformed CD8⁺ cells produce an inhibitoryactivity mediated by a soluble factor capable of passing through amembrane.

[0054] In an embodiment of the invention permanently established CD8⁺transformed cell lines may be generated using a variety of methods. Forexample, CD8⁺ cells prepared from HIV⁺ patients may be transfected withcellular oncogenes such as, for example, ras, src, fos and myc.Alternatively, the CD8⁺ cells may be transfected with oncogenic viralgenes such as EIA of adenovirus, large T of SV40 or middle T of polyomavirus. Additionally, purified CD8⁺ cells may be infected with varioustransforming viruses such as Herpes, SV40, HTLV or adenovirus.

[0055] Once permanently established cell lines are obtained, they may besubjected to limited dilution cloning. The resulting cell clones may beassessed for their ability to produce the CD8⁺ suppressor molecule.

[0056] The permanently established cell lines of the invention aredefined as those CD8⁺ cells producing antiviral activity and those cellscapable of continuous proliferation and propagation in tissue culture.The availability of such transformed cell lines that produce anti-viralactivity will facilitate the purification and characterization of CD8⁺suppressor molecules.

5.4. Purification and Characterization of CD8⁺ Suppressor Molecules

[0057] A CD8⁺ suppressor molecule of the present invention is producedby CD8⁺ cells. In addition, primary cell clones expressing the antiviralactivity have been isolated (see Section 5.2., supra). The CD8⁺antiviral activity may be isolated, e.g., from the conditioned media ofsuch cells or, alternatively, may be extracted from the cells (forexample, in embodiments wherein the CD8⁺ suppressor molecule isexpressed on the cell surface) and subsequently purified to highspecific activity. Purification of CD8⁺ suppressor molecules may beachieved utilizing various procedures and techniques known in the artwhich include but are not limited to chromatography (e.g., reverse phaseliquid, gel permeation, liquid exchange, ion exchange, size exclusion,affinity chromatography), centrifugation, electrophoretic procedures,differential solubility, or by any other standard technique for thepurification of proteins.

[0058] During any protein purification process, the success of theprocess depends on the availability of a reliable assay system formeasuring the presence of the protein of interest. In an embodiment ofthe invention, inhibition of HIV-1 LTR and/or Tat dependent HIVtranscription may used as an indicator of CD8⁺ suppressor activity. Forexample, a recombinant expression vector may be engineered to containthe HIV LTR promoter sequences cloned adjacent to a reporter gene andsuppressor activity may be measured by assaying for reporter geneactivity. Reporter genes that may be used include, but are not limitedto those encoding chloramphenicol acetyltransferase (CAT), fireflyluciferase, human growth hormone, or green fluorescent protein. In theassay system described here, the LTR/reporter gene constructs areco-transfected into an appropriate target cell, such as an appropriateprimary cell or an appropriate cell line, using transfection methodssuch as, for example, calcium phosphate transfection, DEAE-dextrantransfection, electroporation or liposome-mediated transfection. Thetransfected cells may then be used to test for the presence of antiviralactivity. In a specific embodiment described herein, the HIV-LTRsequences were cloned adjacent to the CAT gene, the construct wastransfected into CD4⁺ cells from HIV infected individuals and thepresence of CD8⁺ antiviral activity was determined by measuring CATactivity (FIG. 3 and FIG. 4).

[0059] In another preferred embodiment of the invention, inhibition of asingle cycle pseudotyped virus can be used as an assay system to measurethe presence of a suppressor molecule of interest. For example, a singlecycle pseudotyped virus can be produced which comprises a reportervector having a reporter gene that is expressed during early proviralgene expression. A pseudotyped virus can be engineered, for instance, tocontain a reporter gene in place of an early proviral gene such as theHIV tat, rev or nef gene. In particularly preferred embodiments, thepseudotyped virus contains a reporter gene in place of an HIV nef gene.Reporter genes that may be used include, but are not limited to thoseencoding chloramphenicol acetyltransferase (CAT), firefly luciferase,human growth hormone, or green fluorescent protein (GFP). For exampleand not by way of limitation, in the specific embodiment described inExample 10 below, an Env deficient reporter virus construct containing aluciferase reporter gene in the place of the viral nef gene wascotransfected with a second construct which expressed the product of theviral Env gene, thereby producing a pseudotyped virus which is capableof infecting cells expressing CD4⁺ and an appropriate co-receptor (e.g.,CXCR4 or CCR5) and recapitulating viral replication up to the expressionof early virus genes.

[0060] A known or suspected CD8⁺ suppressor compound, including aprotein of interest in the present invention, can then be screened forsuppressor activity by contacting the compound to a cell, e.g., a CD4⁺cell, after or concurrently with exposing the cell to theabove-described pseudotyped virus and testing for the presence ofantiviral activity by detecting expression and/or activity of thereporter gene. The replication deficient HIV pseudotyped virus of thepresent invention can also be used in screening assays to identifycompounds that suppress viral replication at a particular stage orstages of the virus's replication cycle. For example, Section 10, below,describes exemplary “time of addition” assays which determine that aCD8⁺ suppressor molecule produced by certain cell lines inhibit HIVreplication at a stage of the HIV replication cycle after cell fusionand/or viral entry (i.e., after fusion of the cell and viral membranesand entry of the virus into the cell) and during or immediately prior toexpression of the early proviral genes.

[0061] Time of addition assays of the present invention, including thetime of addition assays described, below, in Section 10, simply comprisesteps of repeatedly infecting a host cell with a replication deficientHIV pseudotyped virus of the invention followed by contacting the hostcell, at different time intervals, with a test compound under conditionssuch that inhibition of HIV replication can occur and measuring activityof the reporter gene of the replication deficient pseudotyped virus. Ahost cell can be infected with a replication deficient pseudotyped virusof the invention by simply contacting the pseudotyped virus to the hostcell under conditions, which are well known in the art, that permitinfection of the host cell by the pseudotyped virus. By observingwhether reporter gene activity is increased or decreased aftercontacting the host cell with a test compound at a certain time intervalafter viral infection, the skilled artisan can identify the stage orstages during which the test compound inhibits HIV replication. Inparticular, inhibition of reporter gene activity after a time intervalassociated with a particular stage in the virus replication cycleindicates that the test compound targets a later stage of the HIVreplication cycle.

[0062] Time intervals after infection that are associated withparticular stages of the HIV replication cycle are already known in theart (see, e.g., Dragic et al., 1996, Nature 381:667-673; Srivastava etal., 1991, J. Virol. 65:3900-3902). Alternatively, however, a skilledartisan can determine such time intervals without undue experimentationby simply performing the time of addition assays of the presentinvention using one or more known inhibitors that target and inhibit HIVreplication at particular, known stages in the replication cycle. Forexample, the time of addition assays described in Section 10, below, arealso performed using particular inhibitors of cell fusion and viralentry (e.g., DP178 and anti-CD4⁺ receptor mAb), reverse transcriptaseinhibitor (e.g., nevirapine) and an inhibitor of early proviral geneexpression (e.g. tat inhibitor) to determine the time intervalsassociated with each of those stages of HIV replication.

[0063] Other compounds that are well known in the art can also be usedto identify the time interval after infection during which a particularstage or stages fo the virus replication cycle occur. For example,inhibitors that block viral entry include peptides and peptide mimetics,referred to herein as anti-fusion peptides, which block membrane fusionevents necessary for viral entry into a host cell. Many such anti-fusionpeptides are well known in the art and include DP178, which is alsoknown in the art as T20 (see, e.g., Bolognesi et al., U.S. Pat. No.5,464,933 which issued on Nov. 7, 1995), DP107, which is also known inthe art as T21 (see, e.g., Wild et al., U.S. Pat. No. 5,656,480 whichissued on Aug. 12, 1997), T1249 and T649 (Barney et al. InternationalPatent Publication No. WO 99/59615 which published on Nov. 25, 1999) toname a few. Sequence search motifs are also known in the art and taught,e.g., in International Patent Publication Nos. WO 94/28920 and WO96/19495, published on Dec. 22, 1994 and Jun. 27, 1996, respectively, bywhich a skilled artisan can readily identify other anti-fusion peptidesthat inhibit membrane fusion-associated events such as HIV infection.

[0064] Antibodies that inhibit viral entry are also known in the art canbe used in time of addition assays of the present invention, e.g., toidentify time intervals associated with viral entry. Such antibodiestypically bind to a receptor, such as the CD4⁺ receptor or aco-receptor, e.g., the CCR5 or CXCR4 chemokine receptor, which isinvolved in or mediates entry of the virus into the host cell.Alternatively, however, monoclonal antibodies that target an HIVprotein, preferably a protein such the HIV envlope glycoprotein (gp120),can also be used to inhibit viral entry into a host cell. Many suchantibodies are already well known in the art, including anti-CD4⁺monoclonal antibody #19 (Endres et al., 1996, Cell 87:745-756).Techniques are also well known to produce antibodies, includingpolyclonal and monoclonal antibodies which target particular proteinssuch as the cell surface receptor and HIV enviope glycoproteinsdescribed above (see, e.g., Harlow and Lane, 1988, Antibodies: ALaboratory Manual, Cold Spring Harbor, N.Y.). Other compounds such asCXCR4 attachment inhibitors, including bicyclams such as AMD3100, SID791, JM3100 and JM2763 (see, e.g., Donzella et al., 1998, Nat. Med.4:72-77), can also be used in time of addition assays of the inventionto inhibit cell entry of HIV virus and thereby identify time intervalsassociated with that stage of the virus replication cycle. Naturalligands for chemokine receptors can also be used in time of additionassay to inhibit cell entry of HIV virus. For example, the CXC stromalcell-derived factor 1 (SDF-1) is a ligand for CXCR4 and can inhibit X4strains of HIV (see, e.g., Oberlin et al., 1996, Nature 382:833-835;Bleul et al., 1996, Nature 382:829-832; Lacey et al., 1997, Proc. Natl.Acad. Sci. U.S.A. 94:9842-9847). Alternatively, the chemokines RANTES,MIP-1α and MIP-β are CCR5 ligands which can inhibit R5 strains of HIV(see, e.g., Cocchi et al., 1995, Science 279:1811-1815; Alkhatib et al.,1996, Science 272:1955-1958; Deng et al., 1996, Nature 381:661-666; andDragic et al., 1996, Nature 381:667-673). Any of these compounds can beused in the time of addition assays of the present invention.

[0065] Time intervals after viral infection that are associated withreverse transcription of the viral RNA genome can also be identified,e.g., by time of addition studies using compounds such as reversetranscriptase inhibitors that block this stage of the viral replicationcycle. Reverse transcriptase inhibitors are well known in the art andinclude both nucleoside and non-nucleoside reverse-transcriptaseanalogs. Non-nucleoside analogs are preferred in the time of additionassays of the present invention and include compounds, such asnevirapine, delavirdine and efavirenz, to name a few. However,nucleoside derivatives, although less preferable, can also be used,including compounds such as 3′azido-3′thymidine (AZT); dideoxyinosine(ddI); 2′,3′-dideoxyadenosine (ddA); 2′,3′-dideoxyguanosin (ddG);2′,3′-dideoxycytidine (ddC); 2′,3′-dideoxythymidine (ddT);2′3′-dideoxy-dideoxythymidine (d4T); and 2′-deoxy-3′-thia-cytosine (3TCor lamivudime). Halogenated nucleoside derivatives may also be usedincluding, for example, 2′3′-dideoxy-2′-fluoronucleosides such as2′,3′-dideoxy-2′-fluroadenosine; 2′,3′-dideoxy-2′-fluoroinosine;2′,3′-dideoxy-2′-fluorothymidine; 2′,3′-dideoxy-2′-fluorocytosine; and2′,3′-dideoxy-2′,3′-didehydro-2′-fluoronucleosides including, but notlimited to2′3′-dideoxy-2′,3′-didehydro-2′-fluorothymidine (Fd4T),2′3′-dideoxy-2′-beta-fluoroadenosine (F-ddA),2′3′-dideoxy-2′-beta-fluoroinosine (F-ddI) and2′,3′-dideoxy-2′-beta-flurocytosine (F-ddC).

[0066] Using standard techniques for protein purification and the assaysystem described above, the CD8⁺ suppressor protein may be purified tohomogeneity. Once purified, the CD8⁺ protein may be subjected tomicrosequencing, using techniques routinely used by those skilled in theart to determine the amino acid sequence of a protein see, CurrentProtocols in Molecular Biology, Ausubel et al., Green PublishingAssociates and Wiley Intersciences, N.Y.) If the CD8⁺ suppressormolecule is blocked at the amino terminus, the protein may be chemicallycleaved or partially enzymatically digested to yield peptide fragmentsthat may be purified and sequenced.

[0067] The purified CD8⁺ protein may be used for production ofantibodies to epitopes of the CD8⁺ protein. Such antibodies include butare not limited to polyclonal and monoclonal antibodies. For productionof antibodies, various host animals may be immunized by injection withthe CD8⁺ protein including but not limited to rabbits, mice, rats etc.Various adjuvants may be used to increase the immunological response,depending on the host species, including but not limited to Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacilli Calmette-Guerin) and Corynebacterium parvum.

[0068] Monoclonal antibodies to CD8⁺ suppressor molecule may be preparedby using any technique which provides for the production of antibodymolecules by continuous cell lines in culture. These include but are notlimited to the hybridoma technique originally described by Kohler andMilstein, (Nature, 1975, 256:495-497), the human B-cell hybridomatechnique (Kosbor et al., 1983, Immunology Today, 4:72; Cote et al.,1983, Proc. Natl. Acad. Sci., 80:2026-2030) and the EBV-hybridomatechnique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96). In addition, techniques developed forthe production of “chimeric antibodies” (Morrison et al., 1984, Proc.Natl. Acad. Sci., 81:6851-6855; Neuberger et al., 1984, Nature,312:604-608; Takeda et al., 1985, Nature, 314:452-454) by splicing thegenes from a mouse antibody molecule of appropriate antigen specificitytogether with genes from a human antibody molecule of appropriatebiological activity can be used.

[0069] Antibody fragments which contain specific binding sites of theCD8⁺ suppressor molecule may be generated by known techniques. Forexample, such fragments include but are not limited to: the F(ab′)₂fragments which can be produced by pepsin digestion of the antibodymolecule and the Fab fragments which can be generated by reducing thedisulfide bridges of the F(ab′)₂ fragments.

[0070] Alternatively, Fab expression libraries may be constructed (Huseet al., 1989, Science, 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificityto the CD8⁺ suppressor molecule.

5.5. Cloning of CD8⁺ Suppressor Molecule

[0071] The present invention relates to methods for cloning of the CD8⁺suppressor molecule. Using methods which are well known to those skilledin the art, recombinant cDNA libraries may be constructed using RNAprepared from cells known to express the CD8⁺ suppressor molecule. ThecDNA libraries may be constructed using a variety of vector systems,including but not limited to, bacteriophage vectors, plasmid vectors ormammalian expression vectors. See, for example, the techniques describedin Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, N.Y. and Current Protocols in MolecularBiology, Green Publishing Associates and Wiley Intersciences, N.Y.

[0072] The recombinant cDNA libraries may be screened using a number ofdifferent techniques. For example, a mixture of degenerateoligonucleotide probes may be designed utilizing the information derivedfrom protein sequencing of the CD8⁺ suppressor protein (see Section 5.3supra). The oligonucleotides may be labeled and used directly to screena cDNA library for clones containing inserts with sequence homology tothe oligonucleotide sequences. Alternatively, the oligonucleotides maybe used as primers in a polymerase chain reaction. The template for thereaction is cDNA obtained by reverse transcription of mRNA prepared fromcells known to express the suppressor activity. The amplified DNAfragment may be labeled and used to screen a library for isolation offull length clones. In another example, an expression library may bescreened immunologically using polyclonal or monoclonal antibodiesdirected against the CD8⁺ suppressor molecule. In yet another embodimentof the invention, a cDNA library may be engineered into a mammalianexpression vector and screened by transfection into the appropriatemammalian cell line followed by assaying for anti HIV suppressoractivity in the tissue culture supernatant. Alternatively, inembodiments wherein the HIV suppressor molecule is or is suspected ofbeing a non-soluble molecule (e.g., embodiments wherein the HIVsuppressor molecule is expressed or suspected of being expressed on thecell surface), the transfected cell can be contacted with an appropriatetarget cell, such as an HIV infected CD4⁺ cell.

[0073] In one preferred embodiment of the invention, a subtracted cDNAlibrary may be constructed using RNA prepared from expressing andnon-expressing clonal CD8⁺ cells. The subtracted library may be screenedusing the LTR/reporter gene assay system. In another preferredembodiment, the subtracted cDNA library may be screened using a singlecycle pseudotyped virus assay system, such as the single cyclepseudotyped virus assay described in Section 5.1 and in Section 10,below.

[0074] A subtracted cDNA library contains cDNA clones corresponding tomRNAs present in one cell type ([+] cell type) that are not present in asecond cell type ([−] cell type). Construction of this type of libraryenriches for cDNA clones of interest and is used in the isolation of acDNA clone corresponding to a particular mRNA where the screeningprocedure is laborious because a specific DNA sequence or antibody isunavailable.

[0075] In an embodiment of the invention a subtractive library may beconstructed using mRNA prepared from the expressing [+] andnon-expressing [−] clonal CD8⁺ cell lines (see Section 5.2., supra . The[+] cDNA is prepared from cells expressing the antiviral activity andoligonucleotide linkers are ligated onto the ends of the cDNA fragmentresulting in endonuclease recognition sites on each end of the cDNAfragment. The [−] cDNA is prepared with blunt ends and digested with arestriction endonuclease that reduces the [−] cDNA fragments to smallblunt ended fragments. The [+] cDNA is then mixed with a 50-fold excessof fragmented [−] cDNA, the DNAs are heated to melt apart thedouble-stranded DNA, and the single stranded DNA is allowed to reanneal.The only [+] cDNA likely to regenerate double stranded fragments withrestriction endonuclease restriction sites at each end are thosesequences for which no complementary [−] fragments were present.Annealed fragments are ligated in an expression vector havingcomplementary cohesive ends. The resulting cDNA library may be screenedusing the LTR/CAT expression system.

[0076] In yet another embodiment of the invention a method forseparating and cloning differentially expressed mRNAs by means ofpolymerase chain reaction may be used to clone a cDNA coding for theCD8⁺ suppressor molecule (Liang et al., 1992, Science 257:967). Such amethod may be utilized using RNA prepared from expressing andnon-expressing CD8⁺ cell lines.

[0077] In addition, gene expression assays using gene expression arraysor microarrays are now practicable for identifying changes in geneexpression patterns between different cells (see, e.g., Schena et al.,1995, Science 270:467-470; Lockhart et al., 1996, Nature Biotechnology14:1674-1680; and Blanchard et al., 1996, Nature Biotechnology 14:1649).Thus, in another, alternative preferred embodiment of the invention,such gene expression arrays or microarrays can be used to compare mRNAexpression patterns in CD8⁺ cells that exhibit HIV suppression activity(e.g., as determined by one of the assays of the present invention) tomRNA expression patterns in CD8⁺ cells that do not exhibit HIVsuppression activity and therefore do not express an HIV suppressormolecule.

5.6. Uses of CD8⁺ Suppressor Molecules

[0078] Currently approved treatments for HIV infection and acquiredimmunodeficiency disease are pharmaceuticals such as dideoxynucleosidesthat target viral reverse transcriptase (i.e. AZT, ddl, ddC). Thoughsome clinical benefit has been demonstrated for these agents, drugresistant viral mutants arise limiting their usefulness. Moreover, theseagents are only effective against de novo infection and do not exert anantiviral effect against chronically infected or latently infectedcells. More effective treatments for HIV infection and AIDS are greatlyneeded.

[0079] The CD8⁺ subclass of T-lymphocytes produce one or more moleculesthat inhibit HIV replication suggesting the potential usefulness of suchmolecules as a therapeutic for treatment of HIV infection and acquiredimmunodeficiency disease. Because of the ability of CD8⁺ suppressormolecules to prevent virus production in cells already infected, theycan also be of use prophylactically in settings such as verticaltransmission of HIV from mother to infant or in acute exposure to HIV.Because the molecules may play a role in maintaining the asymptomaticstate of HIV infected individuals, they can further be of use forclinical staging of disease progression, monitoring the effects ofimmune or biological response modifier therapy and for assessingeffectiveness of certain vaccination protocols.

6. EXAMPLE CD8⁺ Suppressor Activity Inhibits HIV-1 Replication 6.1.Materials and Methods

[0080] Reverse Transcriptase Assays:

[0081] Peripheral blood mononuclear cells (PBMC) were prepared fromfreshly-drawn, anticoagulated blood by standard Ficoll-Hypaque densityseparation. CD4⁺ and CD8⁺ lymphocytes were purified by attachment toanti-CD4⁺ and anti-CD8⁺ microCellector flasks (Applied Immune Sciences)according to the manufacturers recommendations, washed extensively, andcultured for 3 days in medium containing RPMI 1640, 20%, v/v fetalbovine serum (FBS), 50 U/ml recombinant IL-2 (Hoffmann LaRoche, Inc.) 50μg/ml gentamicin sulfate, and 3 μg/ml phytohemagglutinin (PHA, Sigma,Inc.). Cells were removed from the microCellector flasks, aliquots ofthe CD4⁺ and CD8⁺ cell suspensions were analyzed for relative purity byFACS analysis and cell viability was determined by vital dye exclusion.The remaining cell suspensions were cultured for an additional 24 hr inthe same medium as above but lacking PHA. CD4⁺ cells were adjusted to2×10⁶ cells/ml and 100 μl aliquots were cultured in duplicate ortriplicate wells of 96-well microtitre plate with 100 μl of fresh mediumor 100 μl of autologous CD8⁺ cells (adjusted to 4×10⁶ cells/ml), andcultures were incubated at 37° C. in a humidified CO₂ incubator. At 24hr intervals 100 μl aliquots of cultures supernatants were taken,adjusted to 1% Triton X-100 and assayed for reverse transcriptase (RT)activity as described below or frozen at −70° C. until assayed. Thecultures were fed with 100 μl of fresh medium each time supernatantswere harvested. RT activity was assayed by a modification of thepublished methods of Goff et al., and Willey et al. 10 μl of tritonlysate was mixed with 50 μl of a reaction cocktail containing 50 mMTris-HCl. pH 7.8, 75 mM KCl, 2 mM DTT, 5 mM MgCl₂, 5 μg/ml Poly A, 1.5μ/ml OligodT₁₂₋₁₈, 0.05% NP-40, and 10 μCi/ml 32P-TTP, and incubated at37° C. for 90 min. 40-50 μl aliquots of reaction mixtures were spottedonto either DE-81 paper (Whatmann) or onto NA-45 membranes (Schleicher &Schuell) in a minifold sample filtration manifold (Schleicher &Schuell), and the membranes or paper were washed several times with2×SSC (0.3M NaCl, 0.03M NaCitrate), followed by 2×SSC containingBromophenol blue to locate spots. Autoradiography was performed, and themembranes or DE-81 paper counted using a Packard Matrix 9600 Direct BetaCounter. Results presented are the means of duplicate or triplicatewells.

[0082] HIV-1 LTR CAT Constructs:

[0083] The plasmids used in these studies were as follows: 1) pLTR 18,constructed by inserting the Xhol-BamHI LTR-CAT containing fragment ofpU3RIII (Rosen C A, Sodroski J G, Haseltine W A., 1985) into pTZ19R(United States Biochemical) at the Hind III site by blunt-end ligation.Expression of the chloramphenicol acetyl transferase (CAT) reporter genein this vector is under the control of the HIV-1 LTR promoter; 2) pgtat.a tat expression vector under the control of the CMV-IE promoter (MalimM H., Hauber J., Fenrick R., Cullen B R., 1988); 3) pCMVCAT (kindlyprovided by Dr. B. Cullen, Duke University Medical Center), expressionof the CAT reporter gene in this vector is under the control of the sameCMV-IE promoter present in the pgtat vector.

[0084] Transfections and CAT Assays:

[0085] Purified populations of CD4⁺ and CD8⁺ lymphocytes were preparedfrom freshly-drawn anticoagulated blood as described in Section 6.1.1.,except that purified CD4⁺ and CD8⁺ cells were expanded in culture for2-5 days prior to setting up the transfection. To assess effects of CD8⁺cells on HIV-1 LTR or CMV-IE transcriptional activity, CD4⁺ lymphocytes(20×10⁶ cells) were transfected with 10 μg of plasmid (either pLTR 18 orpCMVCAT) by electroporation using a Bio-Rad Gene Pulser. To assesseffects on tat-mediated HIV-1 LTR transcription. 20×10⁶ CD4⁺ lymphocyteswere transfected by electroporation with 2 μg pgtat and 10 μg pLTR 18.The protocol used for the transfections was previously described by Cannet al. (1988, Oncogene 3:123-128). The settings used for electroporationwere 960 μF, 250 V for a single pulse. 4 ml aliquots of CD4⁺ cells infresh medium (1.25×10⁶ cells/ml) from a single transfection werealiquoted into 4 flasks containing either an equal volume of autologousCD4⁺ cell conditioned medium, an equal volume of autologous CD4⁺ cellconditioned medium containing 10×10⁶ non-transfected autologous CD4⁺cells, an equal volume of autologous CD8⁺ cell conditioned medium, or anequal volume of autologous CD8⁺ cell conditioned medium containing10×10⁶ autologous CD8⁺ cells. The volume of each flask was adjusted to10 ml with a combination of fresh medium (RPMI 1640, 10% heatinactivated FBS, 5% IL-2 (Cellular Products, Inc.) and 1% Pen-Strep(Gibco), and either autologous CD4⁺ or CD8⁺ cell conditioned medium sothat the final concentration of conditioned medium in each flask was50%. Cultures were incubated for 48 hr at 37° C. in a humidified CO₂incubator. Cultures were harvested, and CAT activity was determinedessentially as described by Ballard et al. (1992, Proc. Natl. Acad. Sci.U.S.A. 89:1875-1879), except that 1% Triton X-100 was added to the celldisruption buffer which contained 100 mM Tris-HCl, pH 7.8. CAT activitywas not affected by the presence of 1% Triton X-100. Data are plottedfor CAT activity in cultures containing autologous CD8⁺ cells comparedto the activity measured in cultures derived from the same transfectioncontaining autologous CD4⁺ cells. Horizontal lines are drawn to indicatethe means of each population. To test tat-mediated transcription inheterologous CD4⁺ cells a flask containing heterologous CD8⁺ cellconditioned medium and 10×10⁶ heterologous CD8⁺ cells was substituted ineach transfection. Data are plotted for CAT activity in culturescontaining autologous or heterologous CD8⁺ cells compared to theactivity measured in cultures derived from the same transfectioncontaining autologous CD4⁺ cells.

[0086] CAT activity was measured in cell lysates from culturescontaining autologous CD4⁺ cells, cultures containing autologous CD8⁺cells and cultures containing autologous CD8⁺ cells. Each data set froman individual subject was derived from a single transfection. CATactivity is expressed as percent conversion, each assay was based on5×10⁶ transfected CD4⁺ cells.

6.2. Results

[0087] CD8⁺ cells prepared from HIV-1 infected individuals byimmunoaffinity techniques and stimulated with PHA, inhibit HIV-1replication in autologous, infected CD4⁺ cells. The potency of theantiviral effect is striking. When CD8⁺ cells are incubated with CD4⁺cells in a 2:1 ratio, inhibition of viral replication is virtuallycomplete as measured by reverse transcriptase (FIG. 1).

[0088] To investigate the mechanism of CD8⁺ antiviral activity theeffect of CD8⁺ cells on HIV-1 transcription was examined. Autologous andheterologous PBMC-derived CD4⁺ cells were transfected by electroporationwith an HIV-1 LTR CAT construct and a construct that expresses theproduct of the tat gene. CAT activity was measured in the presence ofCD8⁺ cells and as illustrated in FIGS. 3 and 4, CD8⁺ cells from HIV-1infected individuals inhibit tat-mediated HIV-1 LTR transcription inautologous and heterologous CD4⁺ cells. Experiments conducted withsupernatants derived from CD8⁺ cell cultures indicate that a significantfraction of the inhibitory activity can be found in the supernatantindicating that the suppressor activity is secreted by CD8⁺ cells (FIG.5).

7. EXAMPLE Isolation of CD8⁺ Clonal Cells Expressing the Anti-HIV-1Suppressor Molecules 7.1. Materials and Methods

[0089] Establishment of CD8⁺ Cell Clones:

[0090] Peripheral blood mononuclear cells (PBMC) were prepared fromfreshly-drawn, anti-coagulated patient blood by standard Ficoll-Hypaquedensity separation. Twenty million washed PBMC were incubated withanti-CD8⁺ bound magnetic microspheres (Dynal, Inc.) at a bead:cell ratio10:1 in RPMI 1640+1% FCS. After 45′ incubation at 5° C. (with occasionalresuspension), CD8⁺ cell/microsphere conjugates were captured on a rareearth magnet, washed twice, and recaptured. The conjugates weretransferred to a T-25 tissue culture flask containing 10 ml of RPMI1640, 20% v/v FBS. 50 mg/ml of gentamicin sulfate, 3 mg/mlphytohemagglutinin (PHA: Sigma, Inc.) and 50 U/ml recombinantinterleukin 2 (IL-2; Hoffmann LaRoche, Inc.) Conjugates were incubatedat 37° C., 5% CO₂ for 48 hours, after which the microspheres wereremoved by magnetic capture. The remaining CD8⁺ cells in suspension wereanalyzed for relative purity by FACS analysis and cell viability wasdetermined by vital dye exclusion. Suspensions of 10² viable CD8⁺ cellswere subjected to limited dilution cloning as follows. All wells of asingle 96-well round-bottom plate received 10 cells per well, two96-well plates were seeded at 1 cell/well, and five 96-well plates wereseeded at 0.1 cell/well. All wells of all 8 plates subsequently received2×10⁵ irradiated (6000R) heterologous PMBC feeders in the presence of200 ng/ml anti-CD3 monoclonal antibody (12F6) and 100 U/ml of IL-2.Plates were incubated at 37° C., 5% CO₂. At 14d intervals, 100 μL ofcell-free supernatant was removed and discarded. Wells were re-fed with100 μL of fresh media containing 20% FCS, 200 ng/ml 12F6, and 100 U/mlIL-2 containing 10⁵ irradiated (6000R) heterologous PMBC feeders. Wellsexhibiting macroscopic evidence of cellular proliferation were selectedfor stepwise expansion into 48-well and 24-well plates and eventuallyT-25 and T-75 flasks. A 14d re-stimulation cycle was utilized throughoutthe expansion of the clonal populations of CD8⁺ cells. Periodic FACSanalyses were performed using an extensive marker panel. Twenty-fourhours prior to assay, dead feeder cells were removed by Ficoll-Hypaquesedimentation.

[0091] Assay of Transcriptional Inhibition Activity in CD8⁺ Cell Clones:

[0092] CD8⁺ cells clones were assessed for their ability to inhibittat-mediated HIV-1 LTR transcription in autologous B lymphocyte celllines (BLCL) transfected with pgtat and pLTR 18 as follows. Autologous Blymphocyte cell lines were prepared as follows. Peripheral blood wasobtained from HIV-1 infected individuals and PBMCs were prepared byFicoll-Hypaque density gradient separation. Seven to ten million cellswere placed in a T-25 flask (Coaster) in 4 ml of cell culture medium(CCM: RPMI 1640, 20% v/v FBS, 50 μg/ml gentamicin). One ml of EBVsupernatant harvested from a marmoset cell line (B95-8; ATCC) and 10 μgof cyclosporin A were added to the cell suspension. The flasks wereincubated undisturbed at 37° C. in a humidified CO₂ incubator for 3-6weeks. Once a stably transformed BLCL was established it was resuspendedin CCM at a concentration of 3×10⁵ cells/ml. Routine cell culturemaintenance entailed centrifugation and resuspension in new CCM every2-3 days. In this manner, exponential growth was obtained with cellviability routinely 85-90%. Autologous BLCL were cotransfected with 0.1μg pgtat/10⁶ BLCL and 0.5 μg pLTR 18/10⁶ BLCL as described in the legendto FIG. 2. Transfected BLCL were aliquoted (1.5×10⁶ cells) into flaskscontaining either 50% BLCL conditioned medium, 3×10⁶ autologous CD8⁺cells with 50% autologous CD8⁺ cell conditioned medium (experiment 1) or3×10⁶ heterologous CD8⁺ cells with 50% heterologous CD8⁺ cellconditioned medium (experiment 2), or 3×10⁶ autologous CD8⁺ clone 2cells with 50% clone 2 cell conditioned medium, or 3×10⁶ autologous CD8⁺clone 4 cells with 50% clone 4 cell conditioned medium, or 3×10⁶autologous CD8⁺ clone 29 cells with 50% clone 29 cell conditionedmedium. The final volume of each culture was adjusted to 10 ml, andcultures were incubated for 48 hr at 37° C. in a humidified CO₂incubator. Cultures were harvested, and CAT activity was assayed asdescribed in the legend to FIG. 2. Data are presented for CAT activitydetermined in cultures containing CD8⁺ cells or CD8⁺ clones compared tocultures only containing transfected BLCL.

7.2. Results

[0093] Peripheral blood mononuclear cells (PBMC) were prepared fromblood of an HIV-1 infected individual. CD8⁺ cells were immunoaffinitypurified from the patient's blood and the purified cells were subjectedto dilution cloning. The resulting cell clones were assessed for theirability to inhibit HIV-1 LTR transcription in autologous B-lymphocytecell lines transfected with the HIV-1 LTR CAT and tat encodingconstructs. As demonstrated in FIG. 5, the cell clones (clone 2, 4 and29) vary in their ability to inhibit CAT activity. Clone 2 (DU. HL-2)exhibits the greatest inhibitory activity with clone 4 (DU. HL-4) notinhibiting CAT activity to any significant degree.

8. EXAMPLE Oligo Clonal CD8⁺ Lymphocytes From Asymptomatic HIV-InfectedIndividuals Inhibit HIV-1 Replication

[0094] CD8⁺ lymphocytes from asymptomatic HIV-1 infected patients arepotent suppressors of virus production from infected CD4⁺ cells. Ingeneral, studies of HIV-1 suppression have been performed in the contextof bulk CD8⁺ cell cultures. Described below are experiments whereinsuppression by clonal populations of CD8⁺ cell cultures is demonstratedamong the virus suppressive clones derived from an individual patient. Amarked heterogeneity between the cells was evident suggesting that CD8⁺mediated cell virus suppression is oligoclonal in nature.

8.1. Materials and Methods

[0095] Lymphocyte Isolation:

[0096] HIV-1 infected asymptomatic volunteers with CD4⁺ counts >400 wereenrolled for this study. Venous blood was obtained under informedconsent from all volunteers. Peripheral blood mononuclear cells wereprepared from freshly drawn, anticoagulated blood via standardFicoll-Hypaque density separation. CD8⁺ and CD4⁺ lymphocytes weresubsequently purified by attachment to anti-CD8⁺ and CD4⁺ microCellectorT-25 flasks (Applied Immune Sciences, Menlo Park, Calif.) according tomanufacturer recommendations. The cells were cultured in the captureflasks with RPMI-1640 medium containing 20% (v/v) FBS, recombinantinterleukin-2 (IL-2) (100 IU/ml)(Hoffmann La Roche, Inc., Nutley, N.J.),and gentamicin sulfate (50 μg/ml). In addition, mitogenic stimulationwas accomplished with either phytohemagglutinin (PHA [2 μg/ml]; Sigma,St. Louis, Mo.) for CD4⁺ targets or a combination of anti-CD3 (12F6 [100ng/ml]; anti-CD28 (100 ng/ml; Becton-Dickinson) antibodies which wasused for the CD8⁺ effector populations.

[0097] Cells were cultured for three days at 37° in a humidified CO₂incubator. After harvesting, cell viability generally exceeded 95%. Cellpurity of cultures prepared in this manner generally ranged from 87% to95%, as determined by FACS analysis. Bulk populations were harvested,washed and cultured in the same media (lacking PHA or anti-CD3/CD28) for3-5 days. CD8⁺ cells were cloned while CD4⁺ cells were expanded for useas targets in viral inhibition assays.

[0098] CD8⁺ Cell Cloning:

[0099] Bulk CD8⁺ cultures were plated in 96-well plates (Costar,Cambridge Mass.) at a density of 0.5 and 1 cell/well in RPMI-1640 mediumcontaining 20% (v/v) FBS, recombinant interleukin-2 (100 IU/ml),anti-CD3 (100 ng/ml), anti-CD28 (100 ng/ml) and gentamicin sulfate (50μg/ml). A total of 1×10⁵ irradiated (6000 R) allogeneic PBMC/well wereadded as feeders. Wells with cell growth were expanded in 96 or 48-wellplates and restimulated every 10-14 days using the same protocol.

[0100] Viral Suppression Assay:

[0101] Duplicate or triplicate 100 μl aliquots of either heterologous orautologous CD4⁺ cells (1×10⁶ cells ml) were added to wells of a 96 wellmicrotitre plate. For acute infection, CD4⁺ cells from a seronegativeMHC Class I (MHC-I) mismatched donor were inoculated with approximately1×10⁴ TCID₅₀ of HIV_(LAI) (derived from CEM cells) for 1 hour in a 37°C. humidified incubator prior to addition of effectors. For screeningassays, approximately 1-4×10⁵ CD8⁺ cells, within 72 hours of polyclonalrestimulation, were added to the autologous CD4⁺ targets. In titrationexperiments, CD8⁺ clones at the same restimulation interval as in thescreening assays were used as effectors against autologous andheterologous CD4⁺ targets at the following CD8:CD4 ratios: 2.0, 1.0,0.50, 0.25, 0.12. Transwell experiments were performed in 12 welltranswell plates with a membrane pore size of 0.4 μm (Costar). 4×10⁶CD8⁺ cells per clone were cultured on the bottom well in 2 ml of culturemedium. 1×10⁶ acutely infected, heterologous CD4⁺ cells were cultured inthe top chamber in 1 ml of culture medium.

[0102] The CD8⁺/CD4⁺ cell cocultures for both autologous andheterologous assays were incubated for two days. Following the initialtwo day incubation, 100 μl of supernatant from each well was collectedand fresh media was added every other day for the duration of theexperiment. For transwell assays, 1 ml from the bottom well and 300 μlfrom the top well were sampled and replaced with fresh medium.Supernatants harvested from coculture and the top well of the transwellwere retained, added to a clean 96 well microtitre plate and mixed with10 μl per well of a 1% Triton X-100 (Boerhinger Mannheim). Plates werefrozen at −70° C. until assayed (within two weeks).

[0103] Reverse transcriptase activity was assayed according to thepublished methods of Goff et al., 1981, J. Virol. 38:239; and Willey etal., 1988, J. Virol. 62:139 as modified by Chen et al., 1993, AIDS Res.Hum. Retroviruses 9:1079-1086. Briefly, 10 μl of the Triton lysate wasmixed with 50 μl of reaction cocktail consisting of 50 MM Tris-HCL (pH7.8), 75 mM KCI, 2 mM dithiothreitol (DTT), 5 mM MgCl₂, poly(rA), oligo(dT₁₂₋₁₈) [Pharmacia, (1.5 μl/ml)], 0.05% Nonidet P-40 (NP-40)m abd³²P-TTP (10 μCi/ml), and incubated at 37° C. for 90 minutes. Afterincubation, 40 μl aliquots were spotted onto DE-81 paper (Whatman,Clifton, N.J.) in a minifold sample filtrations manifold (Schleicher &Schuell, Keene, N.H.), washed several times with 2×SSC (0.3 M NaCl, 0.03M sodium citrate), followed by 2×SSC containing bromophenol blue to markthe spots. Radioactivity was quantified with a Packard Matrix (Meriden,Conn.) 9600 direct beta counter. Percent inhibition, calculated as[1-(RT ACTIVITY IN CD8⁺CD4⁺COCULTURE)/(RT ACTIVITY IN CD4⁺CULTURE)]X100,was determined over the ten day coculture period.

[0104] Cytoflourimetric Analysis:

[0105] To characterize the phenotype of both suppressive andnonsuppressive clones, three color flow cytometric analysis wasperformed, using different mixtures of monoclonal antibodies. CytostatMoIgG1-PE/MoIgG2a-FITC, CD450PE/CD 14-FITC, CD8-PE/CD3 FITC (Coulter,Hialeah, Fla.) were used as dual color premixes in combination withMouse IgG1-PerCP, CD3-PerCP (Becton Dickinson, San Jose, Calif.)respectively, to determine background fluorescence and lymphocytepurity. Additionally, CD45RO-PE, CD45RA-FITC, CD57-PE, CD28-PE,TCRαβ-FITC, CD38-PE, HLA-DR-FITC (Becton Dickenson), IL-2R-PE (CD25),and S6F1-PE (Coulter) were used with CD8-PerCP. From the three colorcombinations used, percentages for CD8⁺ cells expressing CD45RO+,CD45RA+, CD25+, TCR αβ+, CD38+, HLA-DR+, CD57+, S6F1+, or CD38+/HLA-DR+were determined by using the dual positive population from the two colorhistogram of the appropriate dye combinations.

[0106] In preparation for flow cytometric analysis, the clones werewashed five days after restimulation and incubated for 48 hours in freshmedium. The cells were then washed in 3 ml of Dulbecco'sphosphate-buffered solution (PBS)(GIBCO, Grand Island, N.Y.) andresuspended in RPMI (GIBCO) at a concentration of 10⁶ cells/ml. Next,100 μl of the sample was incubated with 5 μl of the Coulter antibodies(2.5 μl for S6F1) and 10 μl of the Becton Dickinson antibodies in theappropriate combinations for fifteen minutes at room temperature. Thesamples were washed with 3 ml of PBS, resuspended in 250 μl of PBScontaining 1% paraformaldehyde, and held at 4° C. in the dark untilanalysis was performed on a Coulter EPICS Profile II.

8.2. Results

[0107] Clonal Suppression of RT Production:

[0108] The cloning strategy employed for these studies involved initialcapture of CD8⁺ cells from patient PBMC, followed by plating 0.5 or 1cell per well in 96-well plates. The overall cloning efficiency rangedfrom 50-60%. Fifty to seventy-five positive growth wells were randomlyselected for the screening assays of viral suppression.

[0109] Some CD8⁺ clones derived from three patients exhibited strongantiviral activity against autologous and heterologous CD4⁺ lymphocytes.FIG. 7 is a composite showing the effects of representative clones froma typical screening assay. Clones varied in their ability to affect HIVreplication. For example, some clones in culture with autologousnaturally infected CD4⁺ cells as well as heterologous naturally oracutely infected CD4⁺ cells, reduced RT production 95-99% (i.e. S8, B11,and R59), while other clones had little or no effect on RT production(i.e. S6A, B18, R62). Clone B6 was intermediate in the screening assaywith inhibition of 65%.

[0110] Our routine screening assays examine viral suppression at asingle time point on either day 4, 6, or 8 of culture. However, thesuppressive effect can extend throughout a ten day culture interval.FIG. 8 is a composite of the original radiographic data from the microRT assays for two representative clones as well as acutely infected CD4⁺cells alone over the course of a 10-day cocultivation period.Significant suppression of RT signal by clone B11 is evident throughoutthe assay period while the nonsuppressive B18 clone is devoid ofsuppressive activity.

[0111] Of the clones routinely selected from screening, approximately20% were capable of inhibiting RT activity by 75% or greater, whereasonly 4% showed greater than 90% RT inhibition. After screening, cloneswere extensively evaluated for as long as they could be maintained inculture.

[0112] Clonal Suppression in Transwell:

[0113] Further evidence to support a soluble factor based andnon-cytolytic mechanism of HIV suppression by the clones was obtainedwith transwell culture vessels which separated cell populations by a 0.4μM pore-size filter. When CD8⁺ clones were physically separated frominfected CD4⁺ cells, the hierarchy of suppressive ability among theclones was maintained. The addition of the suppressive clone B11(4×10⁶cells) to the bottom well of the culture vessel resulted in significantvirus suppression of acutely infected CD4⁺ cells (1×10⁶ cells) in thetop well (FIG. 9). Conversely, clone B18 showed little inhibition ofvirus replication in the same experiment (FIG. 9). A cocultureexperiment was run in parallel with the transwell experiment. A similarpattern of suppression was evident although suppression was consistentlymore potent in the coculture assays.

[0114] Expression of Cell Surface Markers:

[0115] To more clearly define the subsets of CD8⁺ cells which werecapable of virus suppression, we used dual-color flow cytometry tomeasure surface antigens and RT-PCR to determine the TcR-Vβ expression.The FACS analyses and aforementioned cytokine analyses were performed onthe clones at equivalent times in their activation cycle. Moreover, theday when these analyses were performed corresponds to the day thefunctional suppression analysis was begun. Thus, minor differencesappearing among individual clones are not due to differences in thepost-activation intervals.

[0116] A summary of cell surface marker expression on subsets of CD8⁺cells is as follows: Almost all of the clones expressed elevated levels(>45%) of CD45RO and CD38 but reduced levels (<35%) of CD57 and CD45RA.Expression of the activation markers (HLA-DR, S6F1, CD25, and CD28)appeared highly variable. Expression of the activation markers HLA-DRand S6F1 was significantly higher in the three most suppressive clones,B11, B22.5 and S92, than in clone B6. CD25 expression was elevated(>40%) in three of the four suppressive clones. Also, CD28 expressionwas higher in clone B6 (64%) than in B11 (35%), B22.5 (37%), and S92(16%). Furthermore, B5.5 and B18 expressed reduced (<25%) levels ofHLA-DR, S6F1 and CD25. These data indicated that a diversity ofphenotypic activation markers are displayed within the suppressive andnonsuppressive clonal populations with suppressive clones tending toexpress activation markers to a much higher degree than nonsuppressiveclones. The phenotypes have remained stable for at least three months inculture.

[0117] In addition to activation marker screening, the Vβ region of theTCR was analyzed to check for both clonality and possible TCR repertoirerestriction. Indeed, each clone possessed a single Vβ—indicative of abona fide clone. The variability in Vβ type indicated that there was noclear pattern of Vβ expression among the clones that correlated withsuppressive activity. The only Vβ type which was expressed in more thanone clone, 5.1, appeared in both a suppressive (B6) and a nonsuppressive(B5.5) clone. It should also be noted that 4 of the typed clones werefrom the same individual and no Vβ trends were observed. Collectively,these Vβ and phenotypic data indicate that these CD8⁺ clones withantiviral activity are phenotypically heterogeneous and do not belong toa specific CD8⁺ cell subset.

9. EXAMPLE Generation of Transformed CD8⁺ Suppressor CD8⁺ Cells 9.1.Materials and Methods

[0118] Preparation of CD8⁺ Cells:

[0119] Venous blood was obtained from asymptomatic HIV⁺ patients who hadbeen infected for >7 years. PBMCs were prepared by standardFicoll-Hypaque density separation. CD8⁺ cells were purified by captureon anti-CD8⁺ microCellector flasks (Applied Immune Sciences) accordingto the manufacturer's recommendations, and cultured for 3 days in RPMI1640 medium supplemented with 10% FBS, 20 units/ml recombinantinterleukin 2 (rIL2), penicillin and streptomycin, and 1 μg/mlphytohaemagglutinin (PHA) at 37° C. in a humidified incubator.

[0120] Transformation of CD8⁺ Cells With HVS:

[0121] Three days after PHA activation 2.5×10⁷ CD8⁺ cells from eachpatient were resuspended at 2×10⁶/ml in RPMI 1640 medium supplementedwith 20% FBS, 20 units/ml recombinant IL2, penicillin and streptomycin.An equal volume of HVS 488-77 stock was added to an M.O.I of 0.5. Thecultures were maintained at 37° C. in a gassed CO₂ incubator with aslittle disturbance as possible, refeeding when necessary with freshmedium. After 1 month the viability as measured by erythrosin red dyeexclusion had fallen to 30%. At this point viable cells were recoveredby Ficoll-Hypaque separation. The removal of dead cells was repeated asnecessary over the following 4 weeks, by which time a transformed growthphenotype was evident.

[0122] Virus Suppression Assays:

[0123] Virus suppression assays were performed essentially as previouslydescribed by Chen et al., 1993, AIDS Res. and Hum. Retrovir.9:1079-1086. CD4⁺ target cells were prepared from asymptomatic HIV⁺patients using CD4 microCellector flasks and were further depleted ofCD8⁺ cells by using anti-CD8⁺ antibody coated magnetic beads (Dynal).These CD4⁺ cells were cultured at 2×10⁶/ml in 100 μl aliquots in 96 wellplates. CD8⁺ cells or supernatants from CD8⁺ cell cultures were added atvarying ratios to a final volume of 200 μl. The medium used throughoutwas AIM-V medium supplemented with 20% FBS, 20 units/ml recombinant IL2,penicillin and streptomycin. The plates were incubated at 37° C. in agassed, humidified incubator. At each sampling time point 80 μl of SNwas removed, adjusted to 1% Triton X100, and assayed for reversetranscriptase activity. The RT assay method has been describedpreviously (Goff et al., 1981, J. Virol. 38:239-248; Willey et al. 1988,J. Virol. 62:139-147, Chen et al. 1993, AIDS Res. and Hum. Retrovir. 9).After sampling, the cultures were refed with fresh medium or withconditioned supernatant mixed with fresh medium so as to maintain theoriginal assay conditions. Some of these assays were performed using theTranswell system (Costar) in which typically the CD8⁺ effector cellswere seeded in 1.5 mls of medium into the bottom compartment of a24-well culture plate, and the CD4⁺ target cells were added to the uppercompartment in 500 μl. Sampling was performed from the top compartmentas described above, but refeeding was additionally done at this time byremoving 1 ml of medium from the lower CD8⁺ compartment, and replacingwith 1 ml of fresh medium.

[0124] PCR Analysis of HVS Sequences:

[0125] Total DNA was extracted from 1×10⁶ HVS-transformed CD8⁺ cells andfrom untransformed HIV⁺ CD4⁺ cells by standard phenol/chloroformextraction. DNA equivalent to 3×10⁴ cells was used as the target in PCRreactions employing the following oligonucleotide primer pairs: For theHVS dihydrofolate reductase gene; 5′ GAGAGCTCAAAATCATAACTAGCT 3′(nucleotides 4057-4080 in the HVS genome (Biesinger et al. 1990) and 5′GGTTCTTTTGCTAAACTGTATTGTTGCTG 3′ (4664-4692). For the HVS ORF2 GENE; 5′AGTTCCACACAACTAACTACTAGATGAGAT 3′ (1061-1089) and 5′ATGGCAAGCGAACCGAACCTAAGATATCCA 3′ (1412-1441). The PCR reactionscontained 50 mM KCl, 10 mM Tris-HCl pH8.3, 1.5 mM MgCl₂, 100 μM of eachof dCTP, dGTP, dATP and dTTP, 2.5 ng of each primer, and 2.5 units ofAmplitaq DNA polymerase in a total volume of 100 μl. The thermal cyclingconditions were 6 minutes at 95° C., 30 seconds at 45° C., and 3 minutesat 72° C., PCR products were analyzed on agarose gels containingethidium bromide.

[0126] MHC-Restricted Cellular Cytotoxicity:

[0127] The presence of CD8⁺ cytoxic lymphocytes among the immortalizedcells was detected by standard ⁵¹-Cr release assay. AutologousEpstein-Barr virus immortalized B lymphocytes (BLCL) were used as targetcells after infection with recombinant vaccinia-HIV constructs. Briefly1×10⁶ BLCLs were infected (multiplicity of infection=5:1) for 90 minutesat 37° C. with the following recombinant viruses expressing the E. colilac operon, and the Env, Gag, Pol, and Nef antigens of the HTLV-111Bisolate, respectively: vSC8 (Chakrabarti et al. 1985, Mol. Cell. Biol.5:3403-3409), vPE16 (Earl et al. 1990, J. Virol. 64:2448-2451), vDK1,vCF21 (Flexner et al. 1988, Virol. 166:339-349) and vP1218. Cells wereradiolabeled with 100-200 nCi of sodium chromate (⁵¹-Cr; DuPont,Wilmington, Del.) for 16 hours at 37° and 5% CO₂. The cells weresubsequently washed, counted and plated at a concentration of 5×10³viable cells/well plate. HVS-immortalized CD8⁺ lymphocytes were used aseffectors at the E:T ratios of 100 and 50:1; each E:T was tested intriplicate. Targets plus medium or 0.5% Triton X-100 were used ascontrol for spontaneous (SR) and maximum release (MR) respectively in 4hour-assay. The percentage of specific lysis (%SL) was calculatedaccording to the formula [(cpm experimental release) minus (cpmSR)]/[(cpm SR)]×100. Spontaneous release did not exceed 20% MR.

[0128] The presence of anti-HIV CTL activity was defined as positive ifthe %SL against BLCL expressing HIV antigens was 10% higher than the %SLagainst the control.

[0129] Cloning of CD8⁺ (HVS) Cells:

[0130] The bulk transformed CD8⁺ cells were resuspended in fresh culturemedium (AIM-V medium supplemented with 20% FBS, 20 units/ml recombinantIL2, penicillin and streptomycin) in a 15 ml tube and left to stand for1 hour to allow cell aggregates to sediment. The cell suspension wasthen counted and diluted before plating in 96-well culture dishes at0.25 cells per well. 5×10³ irradiated PBMCs were added to each well asfeeders. The cultures were fed at weekly intervals with fresh medium andexpanded as necessary.

9.2. Results

[0131] Establishment of Transformed CD8⁺ Cell Populations:

[0132] CD8⁺ cells were prepared from several asymptomatic HIV⁺ patients,activated with PHA for 3 days and then exposed to HVS. Afterapproximately 30 days we observed cessation of cell growth and a rapiddecline in cell viability in some of our cultures (patients 1 and 3). Inanother culture (patient 2) we observed continuousactivation-independent growth against a background of cell death,consistent with the expansion of a transformed sub-population (FIG. 10).All the studies described in this work were conducted using laterpassages of the transformed culture from patient 2. The growthproperties of this culture were examined by seeding 5×10⁶ cells into 10mls of AIM-V medium (unsupplemented with FCS), containing varyingconcentrations of rIL2 for approximately 60 hours (FIG. 11). The growthof this population declines if it is moved to serum free medium forextended periods of time (>3 weeks). The experimental results describedin the following sections were performed on later passages of thetransformed culture from patient 2.

[0133] Detection of HVS DNA Sequences in Transformed CD8⁺ Cells:

[0134] Total cell DNA was prepared from the CD8⁺ (HVS) cells 60 daysafter exposure to HVS. PCR was performed using primers corresponding tothe viral dihydrofolate reductase gene and to ORF-2. The latter, alsodesignated STP-C488, has been reported to have transforming andtumor-inducing activity and to be responsible for the viral transformingphenotype (Jung et al. 1991, Proc. Natl. Acad. Sci. USA 89:7051-7055).We obtained PCR products of the predicted sizes using the DNA from thetransformed cells as a target (FIG. 12). When we used an extractprepared from the conditioned medium used to cultivate these transformedcells we saw no HVS PCR signals, indicating that no virus-associated DNAwas present in the medium. This correlates with findings that when thiscell-free conditioned medium was used to overlay a HVS-susceptiblemonolayer of OMK cells for extended periods no viral CPE or plaqueformation is observed. We obtained an upper limit for infections HVS ofless than 0.1 pfu/ml. These observations, taken together, indicate thatthe transformed cells stably contain HVS DNA sequences, but that noinfectious virus is being secreted by these cells.

[0135] Transformed CD8⁺ Cells Are Polyclonal and Activated:

[0136] RNA was prepared from the CD8⁺ (HVS) cells, and cDNA synthesizedand analyzed using PCR primers corresponding to the V_(β) region of theT-cell receptor gene. We found that 22 of the 24 V_(β) families wererepresented in the bulk population (FIG. 13), indicating that ourtransformed CD8⁺ culture is polyclonal. We also analyzed the surfacemarker phenotypes by fluorescence activated cell sorting (FACS) andfound that the predominant markers were: CD8, CD25, CD38, S6F1, CD45RO,CD28 and HLA-DR. No CD4⁺ expression was detectable in the population byFACS analysis. The CD25, CD38 and HLA-DR markers are all typical ofactivated T-cells, the CD25 molecule being the IL-2 receptor. Thepresence of this marker on the surface of the majority of the cells maycorrelate with our observation of an IL2-dependent growth phenotype. TheCD45RO surface marker indicates that the population is primarilycomprised of memory cells.

[0137] Transformed CD8⁺ Cells Do Not Exhibit CTL Activity:

[0138] To ascertain whether the CD8⁺ (HVS) cells contained a significantCTL subpopulation four hour [⁵¹Cr] release CTL assays were conductedemploying as targets EBV transformed BLCL cells infected with vacciniaconstructs expressing the HIV-1 env, gag, pol and nef proteins astargets. Despite using a high ratio of effector to target cells, weobserved no significant HLA-restricted cytolysis compared to thevaccinia vector control.

[0139] Transformed CD8⁺ Cells Secrete a Soluble Factor That InhibitsHIV-1 Replication:

[0140] To compare the inhibitory activity of the HVS-transformed CD8⁺cells with that of primary CD8⁺ cells from the same patient, autologoussuppression assays were set up by isolating CD4⁺ cells from the patientand then cocultivating them with the CD8⁺ cells at defined ratios. Theculture supernatants were sampled at regular intervals and assayed forreverse transcriptase activity. The results (FIG. 14A) indicated thatthe transformed CD8⁺ cells were very similar to the primary CD8⁺ cellsin their ability to inhibit HIV-1 production by the autologous CD4⁺cells. To test whether this observed inhibition was MHC-I restricted, asimilar assay was performed employing CD4⁺ cells from a completely MHC-Imismatched HIV-1 patient. Inhibition that was as potent as that seen inthe autologous assay (FIG. 14B), was observed. The level of suppressionwith a CD8⁺:CD4⁺ ratio of 2:1 approached 98%. These observations,together with the lack of detectable activity in the CTL assays,indicates that the observed inhibition is not due to MHC-I restrictedCTL activity.

[0141] A transwell assay system in which the effector cells areseparated from the target cells by an 0.4 μm membrane was used toinvestigate whether the inhibition of virus production by HIV-1 infectedCD4⁺ cells requires cell-to-cell contact. The results from this assay(FIGS. 15) indicate that, while the most potent suppression is seen withcocultivation, at least part of the inhibitory activity is mediated by asoluble factor capable of passing through the membrane. In addition,experimental results indicated that the conditioned medium from thetransformed CD8⁺ cells can inhibit virus production by the infected CD4⁺cells. The level of inhibition seen is comparable to that in thetranswell experiments. Of note is the observation that the degree ofinhibition increased during the course of the experiment. This may bedue to the fact that the cultures were refed with more conditioned CD8⁺supernatant at each time of sampling.

10. EXAMPLE The CD8⁺ Suppressor Molecule Inhibits HIV After Viral Entry

[0142] Additional studies were conducted to more precisely determine thestage or stages of the HIV replication cycle that is or are susceptibleto inhibition by a CD8⁺ suppressor molecule and/or by CD8⁺ cells. Inparticular, the studies described in this section were designed todetermine whether a juncture exists after the initiation of virusinfection that is resistant to CD8⁺ suppressive effects.

10.1. Materials and Methods

[0143] Preparation and Transformation of CD8⁺ Cells:

[0144] Periopheral blood mononuclear cells (PBMC) were prepared fromfreshly-drawn, anticoagulated venous blood drawn from asymptomaticHIV-positive individuals by standard Ficoll-Hypaque density separation.Cells were activated for 3 days with anti-CD3 (OKT3) and anti-CD28antibodies in AIMV medium (Sigma) Supplemented with 10% FBS, recombinantinterleukin 2 (rIL-2, 20 units per ml), streptomycin (50 μg/ml) andgentamicin (10 μg/ml) at 37° C. in a humidified CO₂ incubator. CD4⁺cells were removed by positive selection with anti-CD4⁺ antibody coatedbeads (Dynal, Lake Success, N.Y.). CD8⁺ cells were further purified bypositive selection with anti-CD8⁺ antibody coated beads (Dynal, LakeSuccess, N.Y.). Beads were removed by DetachaBead (Dynal).

[0145] The CD8⁺ cells of two asymptomatic subjects, displaying the mostpotent activity against an X4 HIV insensitive to β-chemokines in aquantitative assay described hereinbelow were chosen for furthercharacterization. The cells were transformed substantially as describedin Section 5.3, above, and by Lacey et al, 1998, AIDS Res. Hum.Retroviruses 14:521-531, using the herpesvirus saimiri (HVS) subgroup Cstrain 48877 to transform the CD8⁺ cells and create continuous celllines.

[0146] Quantitative CD8⁺ Suppression Assay:

[0147] Peripheral blood mononuclear cells were prepared from freshlydrawn HIV-negative pooled donors and separated by standard FicollHypaque density separation. PBMCs were activated for 2-3 days withanti-CD3 antibodies (OKT3) and anti-CD28 antibodies. CD4⁺ and CD8⁺ wereobtained by negative selection with anti-CD 8+and anti-CD4⁺ coatedbeads, respectively. CD4⁺ cells were centrifuged and resuspended infresh medium at 2.0×10⁵ cell/ml. Duplicate 100 μl aliquoats werecultured in wells of a 96-well microtiter plate containing 20 μl ofserially diluted virus: either QZ4734, a non-syncytia-inducing HIV-1primary isolate (R5), or IIIB, a syncytia-inducing HIV-1 laboratorystrain (X4). Fresh media or CD8⁺ cells at at various effector to targetratios were added in 80 μl. Cultures were incubated at 37° C. in ahumidified CO₂ incubator. At 72 hour intervals, 90 μl of supernatant wasremoved and adjusted to 1% with TritonX-100, and cultures were fed withan equal volume of fresh media. The supernatants were assessed for virusreplication by measuring reverse transcriptase activity according to themethod of Chen et al., 1993, AIDS Res. Hum. Retroviruses 9:1079-1086.

[0148] Creation of Pseudotyped Virus, Infection and Luciferase Assays:

[0149] Pseudotyped viruses were produced by transfection of DNA into thehuman embryonic kidney cell line, 293T, using the modified calciumphosphate method described by Connor et al., 1995, Virology 206:935-944.The virus containing supernatants were harvested three days later. Botha reporter virus DNA (pNL4-3 LUCR⁻E⁻) and an expression vector for anHIV-1 envelope protein, pNL4-3, were cotransfected.

[0150] CD4⁺ primary T-lymphocytes were infected for two hours at 37° C.with 100 μl of pseudotyped virus. The initial cell concentration was1.8×10⁶ cells per ml. After 72 hours, cells were washed with phosphatebuffered saline and lysed in 1× luciferase lysis buffer (Promega)followed by one freeze-thaw cycle. The sample was assayed with thePromega Luciferase Assay System and a luminometer (Lumat LB 9501, EG&GBerthold). In certain experiments, DP178 (20 μg/ml), anti-CD4⁺monoclonal antibody #19 (20 μg/ml), nevirapine (0.2 μM), nelfinavir (0.2μM) or a tat inhibitor (50 μM) were added either simultaneously with thevirus or at timed intervals following infection.

10.2. Results

[0151] Establishment of CD8⁺ Cell Clones Expressing the HIV-1 SuppressorMolecule:

[0152] CD8⁺ T-lymphocytes from ten asymptomatic HIV-positive individualswere screened for inhibition of X4 HIV-1 virus replication. CD8⁺ T-cellsfrom two subjects which exhibiting the most potent activity weresubsequently transformed with Herpesivirus saimiri (HVS) to develop celllines with this effector phenotype according to the methods described byLacey et al., 1998, AIDS Res. Hum. Retroviruses 14:521-531. These celllines, which are referred to herein as DU.JR-HVS and DU.HS-HVS, weredeposited with the American Type Culture Collection (ATCC; Manassas,Va.) on March ##, 2000 as described in Section 11, below, and given theAccession Nos. ### ##### and ### #####, respectively.

[0153] Data from a quantitative CD8⁺ suppression assay with these celllines is shown in FIG. 16. The assay characterizes the CD8⁺ T-cellactivity against the X4 IIIB HIV-1 virus and an R5 HIV-1 primaryisolated referred to as QZ4734. Non-transformed CD8⁺ enriched cells froma pool of normal donors did not significantly alter viral titer ofeither virus. However, the HVS-transformed CD8⁺ cells from anasymptomatic HIV-positive individual potently inhibited infection ofboth the QZ4734 and IIIB viruses.

[0154] In more detail, when transformed CD8⁺ cells were incubated withCD4⁺ cells at a ratio of only 0.1 to 1, the CD8⁺ cells reduced the R5HIV viral titer by greater than two logs. Increasing the ratio to 0.5 to1 completely blocked infection at all virus input levels tested,yielding a lower limit of a three log reduction in R5 HIV-1 titer.Similarly, for X4 HIV-1 virus challenge, incubating CD8⁺ cells with CD4⁺cells at a ratio of 0.5 to 1 blocked replication completely at alllevels examined, causing at least a four log reduction in X4 titer.

[0155] It is interesting to note that X4 HIV-1 isolates are isolateswhich infect T-cell lines using the CXCR4 chemokine receptor, whereas R5HIV-1 isolates infect T-cells using the CCR5 receptor. X4 HIV-1 isolatesare typically recovered late in the course of HIV infection and havebeen shown to be insensitive to β-chemokines (see, e.g., Cocchi et al.,1995, Science 270:1811-1815; Dragic et al., 1996, Nature 381:667-673;Alkhatib et al., 1996, Science 272:1955-1958; Deng et al., 1996, Nature381:661-666; and Cocchi et al, 1996, Nat. Med. 2:1244-1247). Thus, theresults presented here indicate that a CD8⁺ suppressor is inhibiting HIVreplication via a novel, non-cytolytic inhibitory mechanism. As will beappreciated by one skilled in the art, however, the systems and assaysof the present invention can be used to characterize compounds thatsuppress or inhibit HIV by both cytolytic and non-cytolytic mechanisms.

[0156] Single Cycle HIV Infection Assay:

[0157] Because assays that rely on multiple rounds of HIV replicationmight confound such studies, a novel CD8⁺ suppression assay was used tomeasure a single cycle of HIV infection. The assay system utilizes anenvelope-defective luciferase reporter virus (as described by Endres etal., 1996, Cell 87:745-756) complemented in trans with the gene for anHIV-1 envelope protein, NL4-3 env. In this system, the reporter geneserves as a surrogate for the expression of early proviral genes. Thecompletion of viral entry, reverse transcription and proviralintegration is therefore required for expression of the reporter gene.However, expression of the reporter gene is independent of several laterstages in HIV-1 replication, including the expression of late geneproducts, assembly, maturation and budding.

[0158]FIG. 17 illustrates data showing suppression of this pseudotypedreporter virus by CD8⁺ cells. CD8⁺ T-cell effectors prepared asdescribed in Subsection 10.1, above, inhibited infection of thepseudotyped reporter virus in a dose dependent manner, whereas CD8⁺T-cells from HIV-negative donors did not. To determine when infectionhad proceeded past the stage or stages which is or are susceptible tothe CD8⁺ suppressive activity, CD8⁺ T-cells were added to CD4⁺ enrichedT-cells at a ratio of 2 to 1 at various times after exposure of the CD4⁺enriched T-cells to the pseudotyped virus (FIG. 18A). Consistent withthe data presented in FIG. 17, the addition of pooled CD8⁺ T-cells fromHIV-negative donors had little effect on virus replication at each timepoint. However, the CD8⁺ effectors derived from an HIV-positiveindividual completely inhibited viral replication, even when added aslate as six hours post-infection. Even at 24 hours post infection, theCD8⁺ effector cells conferred a log reduction in virus replication thatwas 94.3% inhibition of control infection. At 48 hours, the suppressionwas limited to 77% inhibition of the control infection.

[0159] Investigation of Known HIV Inhibitors:

[0160] Because CD8⁺ cells were shown to have the capacity to block viralinfection at the late time points shown in FIG. 18A, similar “time ofaddition” assays were done using the pseudotype reporter virus tocompare other, known HIV inhibitors which act at known stages in thevirus replication cycle.

[0161] First, studies like the one shown in FIG. 18A were conductedusing two inhibitors, DP178 and anti-CD4⁺ monoclonal antibody (mAb),which are known to inhibit viral entry. Specifically, DP178 is an HIVgp41 peptide mimic which blocks cell fusion events necessary for viralentry into CD4⁺ cells (see Wild et al., 1993, AIDS Res. Hum.Retroviruses 9:1051-1053; and U.S. Pat. No.5,464,933). Likewise, theanti-CD4⁺ monoclonal antibody used in this study, which is referred toas mAb#19 (Connor et al., 1995, Virology 206:935-944) is known to blockthe binding of HIV gp120 to CD4⁺ cells.

[0162] The results of these experiments is shown in FIG. 18B. Wheneither of these two agents was added along with the virus at time zero,viral replication was completely inhibited. However, if two hours passedbetween the time of infection and time of addition of the inhibitor,viral infection occurred at levels that were 23-43% of those obtainedfor controls. Further delaying the addition of these inhibitors until 6hours after infection allowed infection levels to reach 30-61% of thecontrol values (i.e., infection levels in the absence of any inhibitor).These results are in stark contrast to the complete suppression of virusinfection by CD8⁺ cells added at either two or six hours after infection(shown in FIG. 18A), and suggest that HIV-1 entry processes are largelycompleted within six hours of virus exposure.

[0163] Time of addition experiments were also done using nevirapine, anon-nucleoside reverse transcription inhibitor. As can been seen in FIG.19, however, if nevirapine is added as late as 10 hours post infection,viral infection levels are as high as 67% of control levels. Viralinfection levels increase to 85% of control levels if nevirapine is notintroduced to the CD4⁺ cells until 14 hours after virus exposure,suggesting that the process of reverse transcription is complete by10-14 hours post virus exposure.

[0164] Further time of addition experiments were done using an inhibitorof tat described by Hsu et al., 1991, Science 254:1799-1802. The tatgene acts at the level of transcriptional activation. Thus, an HIVinhibitor which targets tat acts during the stages of transcription andearly proviral gene expression. Addition of the tat inhibitor any timefrom zero to 24 hours post infection significantly inhibited viralreplication. Indeed, and as shown in FIG. 19, addition of the tatinhibitor as late as 24 hours post viral exposure resulted in viralreplication levels that are only 46% of control levels.

[0165] Finally, a time of addition experiment was performed usingnelfinavir, a protease inhibitor, as a negative control. As seen in FIG.19, however, the protease inhibitor was ineffectual at all time points.This result is not surprising, since protease activity normally occursat a later stage of the virus replication cycle during virus assembly.Thus, this experiment actually confirmed that the novel pseudotypedreporter virus used here reliably detects only inhibitors that targetearly stages of HIV replication (e.g., during viral entry, reversetranscription and proviral integration).

[0166] The results from these experiments suggest, therefore, that thetarget (or targets) inhibited by the CD8⁺ suppressor molecule is (orare) one(s) which is (or are) active during the latest stages of thepseudotyped viruses life cycle. Possible targets therefore including,but are not limited to, integration of viral DNA, transactivation fromthe proviral state, export of tat and/or rev into the cytoplasm and thenback into the nucleus, and/or tat mediated enhancement of transcription.

11. Deposit of Microorganisms and References Cited

[0167] Deposit of Microorganisms:

[0168] The following microorganisms have been deposited with theAmerican Type Culture Collection, (ATCC), 10801 University Boulevard,Manassa, Va. 20110-2209 on the dates indicated below and in compliancewith the Budapest Treaty on the International Recognition of the Depositof Microorganisms for the Purpose of Patent Proceedure. The depositedmicroorganisms have been assigned the following accession numbers:Microorganism Date of Deposit Accession No. DU. HL-2 March 26, 1993 CRL11310 DU. HL-4 March 26, 1993 CRL 11309 DU. WS-1-CD8(HVS) June 6, 1995CRL 11919 DU.JR-HVS March 14, 2000 ### #### DU.HS-HVS March 14, 2000 #######

[0169] References Cited:

[0170] All references cited herein are incorporated herein by referencein their entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

[0171] Many modifications and variations of this invention can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims along with the full scope ofequivalents to which such claims are entitled. Indeed, variousmodifications of the invention in addition to those described hereinwill become apparent to those skilled in the art from the foregoingdescription and accompanying drawings. Such modifications are intendedto fall within the scope of the appended claims.

[0172] Thus, for example, the present invention is not to be limited inscope by the exemplified embodiments which are intended as illustrationsof single aspects of the invention, and any clones, DNA or amino acidsequences which are functionally equivalent are within the scope of theinvention. The present invention is also not to be limited in scope bythe microorganisms deposited as recited hereinabove, since the depositedembodiments are intended as illustrations of single aspects of theinvention. Any microorganisms which are functionally equivalent aretherefore within the scope of the invention. It is also to be understoodthat all base pair sizes given for nucleotides are approximate and areused for purposes of description.

[0173] The present invention provides a method for detecting a CD8+suppressor molecule that has anti-HIV activity, the method comprisingcontacting a host cell with a replication deficient HIV pseudotypedvirus comprising a reporter gene operatively associated with an HIVpromoter; contacting the host cell with a sample comprising enrichedCD8+ cells or a cell culture of CD8+ cells; and measuring inhibition ofreporter gene activity, wherein inhibition of reporter gene activitycorrelates with anti-activity.

[0174] In a further embodiment, the reporter gene is expressed duringearly proviral gene expression. In another embodiment, the reporter geneis expressed in place of an early proviral gene. In another embodiment,the early proviral gene is a nef gene. In another embodiment, thepseudotyped virus is an env deficient pseudotyped virus. In a furtherembodiment, the pseudotyped virus is produced by a method whichcomprises co-transfecting DNA for said pseudotyped virus with a vectorthat encodes a viral envelope protein. In another further embodiment,the viral envelope protein is an HIV Env protein. In another furtherembodiment, the viral envelope protein is a non-HIV viral envelopeprotein.

[0175] In other embodiments, the reporter gene is a luciferase gene, achloramphenicol acetyltransferase gene, a human growth hormone gene or agreen fluorescent protein gene.

[0176] The present invention also provides a method for detecting a CD8+suppressor molecule that has anti-HIV activity, the method comprisingcontacting a host cell with an env deficient HIV pseudotyped viruscomprising a reporter gene substituted for an HIV nef gene such thatsaid reporter gene is expressed in place of the HIV nef gene; contactingthe host cell with a sample comprising (i) enriched CD8+ cells, (ii) acell culture of CD8+ cells, or (iii) an extract or media componenttherefrom; and (c) measuring inhibition of reporter gene activity,wherein inhibition of reporter gene activity correlates with anti-HIVactivity.

[0177] In further embodiments, the reporter gene is a luciferase gene, achloramphenicol acetyltransferase gene, a human growth hormone gene or agreen fluorescent protein gene.

[0178] The present invention also provides a diagnostic assay formonitoring clinical progression of HIV infection, said diagnostic assaycomprising contacting a host cell with a replication deficient HIVpseudotyped virus comprising a reporter gene operatively associated withan HIV promoter; contacting the host cell with samples from an HIVinfected individual, said samples being collected successively from saidindividual; and measuring inhibition of reporter gene activity when eachsuccessive sample is contacted to the host cell, wherein a decrease inthe inhibition of reporter gene activity when each successive sample iscontacted to the host cell indicates progression of HIV infection.

[0179] In a further embodiment, the reporter gene is expressed duringearly proviral gene expression. In another embodiment, the reporter geneis expressed in place of an early proviral gene. In another embodiment,the early proviral gene is a nef gene. In another embodiment, thepseudotyped virus is an env deficient pseudotyped virus. In a furtherembodiment, the pseudotyped virus is produced by a method whichcomprises co-transfecting DNA for said pseudotyped virus with a vectorthat encodes a viral envelope protein. In another further embodiment,the viral envelope protein is an HIV Env protein. In another furtherembodiment, the viral envelope protein is a non-HIV viral envelopeprotein.

[0180] In other further embodiments, the reporter gene is a luciferasegene, a chloramphenicol acetyltransferase gene, a human growth hormonegene or a green fluorescent protein gene.

[0181] The present invention also provides a diagnostic assay formonitoring clinical progression of HIV infection, said diagnostic assaycomprising contacting a host cell with an env deficient HIV pseudotypedvirus comprising a reporter gene substituted for an HIV nef gene suchthat said reporter gene is expressed in place of the HIV nef gene;contacting the host cell with samples from an HIV infection individual,said samples being collected successively from said individual; andmeasuring inhibition of reporter gene activity when each successivesample is contacted to the host cell, wherein a decrease in theinhbition of reporter gene activity when each successive sample iscontacted to the host cell indicates progression of HIV infection.

[0182] In further embodiments, the reporter gene is a luciferase gene, achloramphenicol acetyltransferase gene, a human growth hormone gene or agreen fluorescent protein gene.

[0183] The present invention also provides a method for detecting acompound that inhibits HIV replication after a particular stage ofreplication, said method comprising contacting a host cell with areplication deficient HIV pseudotyped virus comprising a reporter geneoperatively associated with an HIV promoter; contacting the host cellwith a test compound at successive time intervals; and measuringreporter activity at each of said successive time intervals, whereininhibition of reporter activity at a time interval after the particularstage of replication indicates that the test compound inhibits HIVreplication after said particular stage.

[0184] The method of claim 27, wherein the reporter gene is expressedduring early proviral gene expression.

[0185] In a further embodiment, the reporter gene is expressed duringearly proviral gene expression. In another embodiment, the earlyproviral gene is a nef gene. In another embodiment, the pseudotypedvirus is an env deficient pseudotyped virus. In a further embodiment,the pseudotyped virus is produced by a method which comprisesco-transfecting DNA for said pseudotyped virus with a vector thatencodes a viral envelope protein. In a further embodiment, theparticular stage of replication is viral entry.

[0186] In another embodiment, the method further comprises contacting adifferent host cell with said HIV pseudotyped virus; contacting thedifferent host cell with a viral entry inhibitor at successive timeintervals; and measuring inhibition of reporter activity at each of saidtime intervals, wherein those time intervals at which reporter activityis inhibited correspond to time intervals of viral entry. In a furtherembodiment, the viral entry inhibitor is an anti-fusion peptide. Inanother embodiment, the anti-fusion peptide is DP107, DP178, T1249 orT649.

[0187] In another embodiment, the viral entry inhibitor is an antibodythat disrupts the interaction between a CD4+ cell surface receptor and aviral envelope protein. In a further embodiment, the antibody is amonoclonal antibody that specifically binds to the CD4+ receptor. Inanother embodiment, the particular stage of replication is reversetranscription.

[0188] In another embodiment, the method further comprises contacting adifferent host cell with said HIV pseudotyped virus; contacting thedifferent host cell with a reverse transcription inhibitor at successivetime intervals; and measuring inhibition of reporter activity at each ofsaid time intervals, wherein those time intervals at which reporteractivity is inhibited correspond to time intervals of reversetranscription.

[0189] In a further embodiment, the reverse transcription inhibitor is anon-nucleoside reverse transcriptase inhibitor. In one embodiment, thereverse transcriptase inhibitor is nevirapine. In another embodiment,the particular stage of replication is early virus gene expression.

[0190] In another embodiment, the method further comprises contacting adifferent host cell with said HIV pseudotyped virus; contacting thedifferent host cell with an inhibitor of early virus gene expression atsuccessive time intervals; and measuring inhibition of reporter activityat each of said time intervals, wherein those time intervals at whichreporter activity is inhibited correspond to time intervals of earlyvirus gene expression. In a further embodiment, the inhibitor of earlyvirus gene expression is a Tat inhibitor.

[0191] The present invention also provides a method for obtaining apreparation containing a CD8+ suppressor molecule, said methodcomprising collecting conditioned media from cells expressing the CD8+suppressor molecule; fractionating media components of said conditionedmedia; and identifying a fraction having CD8+ suppressor activity by amethod comprising (i) contacting a host cell with a replicationdeficient pseudotyped HIV virus comprising a reporter gene operativelyassociated with an HIV promoter, (ii) contacting the host cell with afractionated media component of said conditioned media, and (iii)measuring inhibition of reporter activity; wherein inhibition ofreporter activity correlates with a fraction containing CD8+ suppressoractivity. In a further embodiment, the reporter gene is expressed duringearly proviral gene expression.

[0192] The present invention also provides a method for obtaining apreparation containing a CD8+ suppressor molecule, said methodcomprising preparing an extract from a cell or cell line expressing theCD8+ suppressor molecule; fractionating components of said extract; andidentifying a fraction having CD8⁺ suppressor activity by a methodcomprising (i) contacting a host cell with a replication deficientpseudotyped HIV virus comprising a reporter gene operatively associatedwith an HIV promoter, (ii) contacting the host cell with a fractionatedcomponent of said extract, and (iii) measuring inhibition of reporteractivity; wherein inhibition of reporter activity correlates with afraction containing a CD8+ suppressor molecule. In a further embodiment,the reporter gene is expressed during early proviral gene expression.

[0193] In one embodiment, the reporter gene is expressed in place of anearly proviral gene. In a further embodiment, the early proviral gene isa nef gene.

[0194] In another embodiment, the pseudotyped virus is an env deficientpseudotyped virus. In a further embodiment, the pseudotyped virus isproduced by a method which comprises co-transfecting DNA for saidpseudotyped virus with a vector that encodes a viral envelope protein.In one embodiment, the viral envelope protein is an HIV Env protein. Inanother embodiment, the viral envelope protein is a non-HIV envelopeprotein. In another embodiment, the conditioned media or extract isprepard from a lymphocyte cell clone that expresses a CD8+ suppressormolecule that inhibits HIV replication.

[0195] The present invention also provides a method for isolating arecombinant cDNA clone encoding a CD8+ suppressor molecule that inhibitsHIV replication, said method comprising constructing a cDNA expressionlibrary using mRNA prepared from CD8+ T-lymphocytes that express a CD8+suppressor molecule; and screening cDNA products from said cDNAexpression library using a method comprising, for each of said cDNAproducts (i) contacting a host cell with a replication deficient HIVpseudotyped virus comprising a reporter gene operatively associated withan HIV promoter, (ii) contacting the host cell with a sample comprisinga cDNA product from the cDNA expression library, and (iii) measuringinhibition of reporter gene activity; wherein inhibition of reportergene activity indicates that a recombinant cDNA clone encodes asuppressor molecule that inhibits HIV.

[0196] In a further embodiment, the reporter gene is expressed duringearly proviral gene expression. In another embodiment, the reporter geneis expressed in place of an early proviral gene. In another embodiment,the early proviral gene is a nef gene. In another embodiment, thepseudotyped virus is an env deficient pseudotyped virus. In a furtherembodiment, the pseudotyped virus is produced by a method whichcomprises co-transfecting DNA for said pseudotyped virus with a vectorthat encodes a viral envelope protein. In another further embodiment,the viral envelope protein is an HIV Env protein. In another furtherembodiment, the viral envelope protein is a non-HIV envelope protein.

[0197] In another embodiment, the method further comprises, before saidscreening step, a step of enriching the cDNA library by eliminatingclones that hybridize to cDNAs prepared from mRNA of lymphocytes that donot express a CD8+ suppressor molecule.

[0198] The present invention also provides a permanently establishedlymphocyte cell line that expresses a CD8+ protein on the cell surfaceand expresses a CD8+ suppressor molecule that inhibits HIV replication,wherein said CD8+ suppressor molecule is a non-cytolytic molecule. Inone embodiment, the cell line is the cell line identified as DU.JR-HVS(ATCC Accession No. ### #####). In another embodiment, the cell line isthe cell line identified as DU.HS-HVS (ATCC Accession No. ### #####).

What is claimed is:
 1. A method for detecting a CD8⁺ suppressor moleculethat has anti-HIV-1 activity, the method comprising: (a) contacting ahost cell with a replication deficient HIV pseudotyped virus comprisinga reporter gene operatively associated with an HIV promoter; (b)contacting the host cell with: (i) a sample comprising enriched CD8⁺cells; or (ii) a sample comprising a cell culture of CD8⁺ cells; or(iii) an extract or media component from (i) or (ii); and (c) measuringreporter gene activity, wherein inhibition of reporter gene activityindicates anti-HIV-1 activity.
 2. The method of claim 1, wherein thereporter gene is expressed during early proviral gene expression.
 3. Themethod of claim 2, wherein the reporter gene is expressed in place of anearly proviral gene.
 4. The method of claim 3, wherein the earlyproviral gene is a nef gene.
 5. The method of claim 1, wherein thepseudotyped virus is an env deficient pseudotyped virus.
 6. The methodof claim 5, wherein the pseudotyped virus is produced by a methodcomprising co-transfecting DNA for the pseudotyped virus with a vectorthat encodes a viral envelope protein.
 7. The method of claim 6, whereinthe viral envelope protein is an HIV Env protein.
 8. The method of claim6, wherein the viral envelope protein is a non-HIV viral envelopeprotein.
 9. The method of claim 1, wherein the reporter gene is aluciferase gene, a chloramphenicol acetyltransferase gene, a growthhormone gene, or a fluorescent protein gene.
 10. The method of claim 9,wherein the reporter gene is a luciferase gene.
 11. A method fordetecting a CD8⁺ suppressor molecule that has anti-HIV-1 activity, saidmethod comprising: (a) contacting a host cell with an env deficient HIVpseudotyped virus comprising a reporter gene substituted for an HIV nefgene such that said reporter gene is expressed in place of the HIV nefgene; (b) contacting the host cell with: (i) a sample comprisingenriched CD8⁺ cells; or (ii) a sample comprising a cell culture of CD8⁺cells; or (iii) an extract or media component from (i) or (ii); and (c)measuring reporter gene activity, wherein inhibition of reporter geneactivity indicates anti-HIV-1 activity.
 12. The method of claim 11,wherein the reporter gene is a luciferase gene, a chloramphenicolacetyltransferase gene, a growth hormone gene, or a fluorescent proteingene.
 13. The method of claim 12, wherein the reporter gene is aluciferase gene.
 14. A diagnostic assay for monitoring clinicalprogression of HIV infection, the diagnostic assay comprising: (a)contacting a host cell with a replication deficient HIV pseudotypedvirus comprising a reporter gene operatively associated with an HIVpromoter; (b) contacting the host cell with samples from an HIV infectedindividual, wherein the samples are collected from the individual at oneor more time intervals; and (c) measuring reporter gene activity at oneor more time intervals, wherein an increase in reporter gene activityindicates progression of HIV infection.
 15. The method of claim 14,wherein the reporter gene is expressed during early proviral geneexpression.
 16. The method of claim 15, wherein the reporter gene isexpressed in place of an early proviral gene.
 17. The method of claim16, wherein the early proviral gene is a nef gene.
 18. The method ofclaim 16, wherein the pseudotyped virus is an env deficient pseudotypedvirus.
 19. The method of claim 18, wherein the pseudotyped virus isproduced by a method comprising co-transfecting DNA for said pseudotypedvirus with a vector that encodes a viral envelope protein.
 20. Themethod of claim 19, wherein the viral envelope protein is an HIV Envprotein.
 21. The method of claim 19, wherein the viral envelope proteinis a non-HIV viral envelope protein.
 22. The method of claim 14, whereinthe reporter gene is a chloramphenicol acetyltransferase gene, aluciferase gene, a growth hormone gene, or a fluorescent protein gene.23. The method of claim 22, wherein the reporter gene is a luciferasegene.
 24. A diagnostic assay for monitoring clinical progression of HIVinfection, the diagnostic assay comprising: (a) contacting a host cellwith an env deficient HIV pseudotyped virus comprising a reporter genesubstituted for an HIV nef gene such that said reporter gene isexpressed in place of the HIV nef gene; (b) contacting the host cellwith samples from an HIV infection individual, wherein the samples arecollected from the individual at one or more time intervals; and (c)measuring reporter gene activity at one or more time intervals, whereinan increase in reporter gene activity indicates progression of HIVinfection.
 25. The method of claim 24, wherein the reporter gene is achloramphenicol acetyltransferase gene, a luciferase gene, a growthhormone gene, or a fluorescent protein gene.
 26. The method of claim 25,wherein the reporter gene is a luciferase gene.
 27. A method fordetecting a compound that suppresses HIV-1 replication, the methodcomprising: (a) contacting a host cell with a replication deficient HIVpseudotyped virus comprising a reporter gene operatively associated withan HIV promoter; (b) contacting the host cell with the compound at oneor more time intervals; and (c) measuring reporter gene activity at oneor more time intervals, wherein inhibition of reporter gene activity atone or more time intervals indicates that the compound suppresses HIV-1replication.
 28. The method of claim 27, wherein the reporter gene isexpressed during early proviral gene expression.
 29. The method of claim28, wherein the reporter gene is expressed in place of an early proviralgene.
 30. The method of claim 29, wherein the early proviral gene is anef gene.
 31. The method of claim 27, wherein the pseudotyped virus inan env deficient pseudotyped virus.
 32. The method of claim 31, whereinthe pseudotyped virus is produced by a method comprising co-transfectingDNA for the pseudotyped virus with a vector that encodes a viralenvelope protein.
 33. The method of claim 32, wherein the viral envelopeprotein is an HIV Env protein.
 34. The method of claim 32, wherein theviral envelope protein is a non-HIV envelope protein.
 35. The method ofclaim 27, wherein suppression of HIV-1 is at a stage of viral entry. 36.The method of claim 35 further comprising the steps of: (a) contacting adifferent host cell with the HIV pseudotyped virus; (b) contacting thedifferent host cell with a viral entry inhibitor at one or more timeintervals; and (c) measuring reporter gene activity at one or more timeintervals; wherein the time intervals at which reporter gene activity isinhibited correspond to time intervals of viral entry.
 37. The method ofclaim 36, wherein the viral entry inhibitor is an anti-fusion peptide.38. The method of claim 37, wherein the anti-fusion peptide is DP107,DP178, T1249 or T649.
 39. The method of claim 36, wherein the viralentry inhibitor is an antibody that disrupts the interaction between aCD4⁺ cell surface receptor and a viral envelope protein.
 40. The methodof claim 39, wherein the antibody is a monoclonal antibody thatspecifically binds to the CD4⁺ receptor.
 41. The method of claim 27,wherein suppression of HIV-1 is at a stage of reverse transcription. 42.The method of claim 41 further comprising the steps of: (a) contacting adifferent host cell with the HIV pseudotyped virus; (b) contacting thedifferent host cell with a reverse transcription inhibitor at one ormore time intervals; and (c) measuring reporter gene activity at one ormore time intervals; wherein the time intervals at which reporter geneactivity is inhibited correspond to time intervals of reversetranscription.
 43. The method of claim 42, wherein the reversetranscription inhibitor is a non-nucleoside reverse transcriptaseinhibitor.
 44. The method of claim 43, wherein the reverse transcriptaseinhibitor is nevirapine.
 45. The method of claim 27, wherein suppressionof HIV-1 is at a stage of early virus gene expression.
 46. The method ofclaim 45 further comprising the steps of: (a) contacting a differenthost cell with the HIV pseudotyped virus; (b) contacting the differenthost cell with an inhibitor of early virus gene expression at one ormore time intervals; and (c) measuring reporter gene activity at one ormore time intervals, wherein the time intervals at which reporter geneactivity is inhibited correspond to time intervals of early virus geneexpression.
 47. The method of claim 46, wherein the inhibitor of earlyvirus gene expression is a Tat inhibitor.